Sample records for active site formation

  1. Carbinolamine Formation and Dehydration in a DNA Repair Enzyme Active Site

    PubMed Central

    Dodson, M. L.; Walker, Ross C.; Lloyd, R. Stephen

    2012-01-01

    In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics–molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water–independent enzyme–catalyzed reaction had a bias–corrected Jarzynski–average barrier height of approximately for the carbinolamine formation reaction and ) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately in the forward (formation) reaction and for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water–independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N–terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site

  2. A DFT Study of Tungsten-Methylidene Formation on a W/ZSM-5 Zeolite: The Metathesis Active Site.

    PubMed

    Maihom, Thana; Probst, Michael; Limtrakul, Jumras

    2015-10-26

    Tungsten-methylidene formation from ethene on either the W(IV) , W(V) , or W(VI) active sites of a W/ZSM-5 zeolite is investigated by using the M06-L functional. The reaction is assumed to proceed in two steps; the first step is the [2+2] cycloaddition between ethene and the W-O active site to form an oxametallacycle intermediate. The intermediate is then decomposed to produce the W-methylidene active site from the metathesis reaction. The overall activation barrier of the reaction on W(VI) (27.3 kcal mol(-1) ) is considerably lower than the ones for W(IV) and W(V) (69.4 and 37.1 kcal mol(-1) , respectively). Moreover, the reaction involving the W(VI) site also stabilizes intermediates and products to a larger extent than the ones on the W(IV) and W(V) sites. As a result, we have demonstrated that the reaction of the W-methylidene metathesis active site is both kinetically and thermodynamically favored to occur on the W(VI) active site of the zeolite. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Formation of RNA oligomers on montmorillonite: site of catalysis

    NASA Technical Reports Server (NTRS)

    Ertem, G.; Ferris, J. P.

    1998-01-01

    Certain montmorillonites catalyze the self condensation of the 5'-phosphorimidazolide of nucleosides in pH 8 aqueous electrolyte solutions at ambient temperatures leading to formation of RNA oligomers. In order to establish the nature of the sites on montmorillonite responsible for this catalytic activity, oligomerization reactions were run with montmorillonites which had been selectively modified (I) at the edges by (a) fluoride treatment, (b) silylation, (c) metaphosphate treatment of the anion exchange sites (II) in the interlayer by (a) saturation with quaternary alkylammonium ions of increasing size, (b) aluminum polyoxo cations. High pressure liquid chromatography, HPLC, analysis of condensation products for their chain lengths and yields indicated that modification at the edges did not affect the catalytic activity to a significant extent, while blocking the interlayer strongly inhibited product formation.

  4. Observing the formation of ice and organic crystals in active sites

    PubMed Central

    Campbell, James M.; Meldrum, Fiona C.; Christenson, Hugo K.

    2017-01-01

    Heterogeneous nucleation is vital to a wide range of areas as diverse as ice nucleation on atmospheric aerosols and the fabrication of high-performance thin films. There is excellent evidence that surface topography is a key factor in directing crystallization in real systems; however, the mechanisms by which nanoscale pits and pores promote nucleation remain unclear. Here, we use natural cleavage defects on Muscovite mica to investigate the activity of topographical features in the nucleation from vapor of ice and various organic crystals. Direct observation of crystallization within surface pockets using optical microscopy and also interferometry demonstrates that these sharply acute features provide extremely effective nucleation sites and allows us to determine the mechanism by which this occurs. A confined phase is first seen to form along the apex of the wedge and then grows out of the pocket opening to generate a bulk crystal after a threshold saturation has been achieved. Ice nucleation proceeds in a comparable manner, although our resolution is insufficient to directly observe a condensate before the growth of a bulk crystal. These results provide insight into the mechanism of crystal deposition from vapor on real surfaces, where this will ultimately enable us to use topography to control crystal deposition on surfaces. They are also particularly relevant to our understanding of processes such as cirrus cloud formation, where such topographical features are likely candidates for the “active sites” that make clay particles effective nucleants for ice in the atmosphere. PMID:27994140

  5. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    DOE PAGES

    Wang, Yanggang; Mei, Donghai; Glezakou, Vassiliki Alexandra; ...

    2015-03-04

    Ab initio Molecular Dynamics simulations and static Density Functional Theory calculations have been performed to investigate the reaction mechanism of CO oxidation on Au/CeO 2 catalyst. It is found that under reaction condition CO adsorption significantly labializes the surface atoms of the Au cluster and leads to the formation of isolated Au+-CO species that resides on the support in the vicinity of the Au particle. In this context, we identified a dynamic single-atom catalytic mechanism at the interfacial area for CO oxidation on Au/CeO 2 catalyst, which is a lower energy pathway than that of CO oxidation at the interfacemore » with the metal particle. This results from the ability of the single atom site to strongly couple with the redox properties of the support in a synergistic manner thereby lowering the barrier for redox reactions. We find that the single Au+ ion, which only exists under reaction conditions, breaks away from the Au cluster to catalyze CO oxidation and returns to the Au cluster after the catalytic cycle is completed. Generally, our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in a catalytic process.« less

  6. OceanSITES format and Ocean Observatory Output harmonisation: past, present and future

    NASA Astrophysics Data System (ADS)

    Pagnani, Maureen; Galbraith, Nan; Diggs, Stephen; Lankhorst, Matthias; Hidas, Marton; Lampitt, Richard

    2015-04-01

    initiative has always been truly international, and in Europe the first project to include OceanSITES as part of its outputs was ANIMATE(2002-2005), where 3 moorings and 5 partners shared equipment, methods and analysis effort and produced their final outputs in OceanSITES format. Subsequent European projects, MERSEA(2004-2008) and EuroSITES (2008-2011) built on that early success and the current European project FixO3 encompasses 23 moorings and 29 partners, all of whom are committed to producing data in OceanSITES format. The global OceanSITES partnership continues to grow; in 2014 the Australian Integrated Marine Observing System ( IMOS) started delivering data to the OceanSITES FTP, and files and India, South Korea and Japan are also active members of the OceanSITES community. As illustrated in figure 1 the OceanSITES sites cover the entire globe, and the format has now matured enough to be taken up by other user groups. GO-SHIP, a global, ship-based hydrographic program, shares technical management with OceanSITES through JCOMMOPS, and has its roots in WOCE Hydrography. This program complements OceanSITES and directly contributes to the mooring data holdings by providing repeated CTD and bottle profiles at specific locations. GO-SHIP hydrographic data adds a source of timeseries profiles and are provided in the OceanSITES file structure to facilitate full data interoperability. GO-SHIP has worked closely with the OceanSITES program, and this interaction has produced an unexpected side benefit - all data in the GO-SHIP database will be offered the robust and CF-compliant OceanSITES format beginning in 2015. The MyOcean European ocean monitoring and forecasting project has been in existence since 2009, and has successfully used the OceanSITES format as a unifying paradigm. MyOcean daily receives hundreds of data files from across Europe, and distributes the data from drifter buoys, moorings and tide gauges in OceanSITES format. These in-situ data are essential for both

  7. Active star formation in NGC 2264

    NASA Technical Reports Server (NTRS)

    Schwartz, P. R.; Thronson, H. A., Jr.; Odenwald, S. F.; Glaccum, W.; Loewenstein, R. F.; Wolf, G.

    1985-01-01

    The region of NGC 2264 near the cone nebula is the site of active star formation in a rotating ring seen nearly edge on as a two lobed source. Allen's infrared source (IRS 1) surrounds a B3V star still embedded in the southern lobe of the cloud. The northern lobe, IRS 2, also probably contains young stars.

  8. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

    PubMed

    Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

    2017-08-30

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

  9. Active site formation mechanism of carbon-based oxygen reduction catalysts derived from a hyperbranched iron phthalocyanine polymer.

    PubMed

    Hiraike, Yusuke; Saito, Makoto; Niwa, Hideharu; Kobayashi, Masaki; Harada, Yoshihisa; Oshima, Masaharu; Kim, Jaehong; Nabae, Yuta; Kakimoto, Masa-Aki

    2015-01-01

    Carbon-based cathode catalysts derived from a hyperbranched iron phthalocyanine polymer (HB-FePc) were characterized, and their active-site formation mechanism was studied by synchrotron-based spectroscopy. The properties of the HB-FePc catalyst are compared with those of a catalyst with high oxygen reduction reaction (ORR) activity synthesized from a mixture of iron phthalocyanine and phenolic resin (FePc/PhRs). Electrochemical measurements demonstrate that the HB-FePc catalyst does not lose its ORR activity up to 900°C, whereas that of the FePc/PhRs catalyst decreases above 700°C. Hard X-ray photoemission spectra reveal that the HB-FePc catalysts retain more nitrogen components than the FePc/PhRs catalysts between pyrolysis temperatures of 600°C and 800°C. This is because the linked structure of the HB-FePc precursor has high thermostability against nitrogen desorption. Consequently, effective doping of active nitrogen species into the sp (2) carbon network of the HB-FePc catalysts may occur up to 900°C.

  10. Different properties of skin of different body sites: The root of keloid formation?

    PubMed

    Butzelaar, Liselotte; Niessen, Frank B; Talhout, Wendy; Schooneman, Dennis P M; Ulrich, Magda M; Beelen, Robert H J; Mink van der Molen, Aebele B

    2017-09-01

    The purpose of this study was to examine extracellular matrix composition, vascularization, and immune cell population of skin sites prone to keloid formation. Keloids remain a complex problem, posing esthetical as well as functional difficulties for those affected. These scars tend to develop at anatomic sites of preference. Mechanical properties of skin vary with anatomic location and depend largely on extracellular matrix composition. These differences in extracellular matrix composition, but also vascularization and resident immune cell populations might play a role in the mechanism of keloid formation. To examine this hypothesis, skin samples of several anatomic locations were taken from 24 human donors within zero to 36 hours after they had deceased. Collagen content and cross-links were determined through high-performance liquid chromatography. The expression of several genes, involved in extracellular matrix production and degradation, was measured by means of real-time PCR. (Immuno)histochemistry was performed to detect fibroblasts, collagen, elastin, blood vessels, Langerhans cells, and macrophages. Properties of skin of keloid predilections sites were compared to properties of skin from other locations (nonpredilection sites [NPS]). The results indicated that there are site specific variations in extracellular matrix properties (collagen and cross-links) as well as macrophage numbers. Moreover, predilection sites (PS) for keloid formation contain larger amounts of collagen compared to NPS, but decreased numbers of macrophages, in particular classically activated CD40 positive macrophages. In conclusion, the altered (histological, protein, and genetic) properties of skin of keloid PS may cause a predisposition for and contribute to keloid formation. © 2017 by the Wound Healing Society.

  11. Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal.

    PubMed

    Jain, Rinku; Hao, Bing; Liu, Ren-Peng; Chan, Michael K

    2005-04-06

    E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.

  12. Heterogeneous Electrocatalyst with Molecular Cobalt Ions Serving as the Center of Active Sites.

    PubMed

    Wang, Jiong; Ge, Xiaoming; Liu, Zhaolin; Thia, Larissa; Yan, Ya; Xiao, Wei; Wang, Xin

    2017-02-08

    Molecular Co 2+ ions were grafted onto doped graphene in a coordination environment, resulting in the formation of molecularly well-defined, highly active electrocatalytic sites at a heterogeneous interface for the oxygen evolution reaction (OER). The S dopants of graphene are suggested to be one of the binding sites and to be responsible for improving the intrinsic activity of the Co sites. The turnover frequency of such Co sites is greater than that of many Co-based nanostructures and IrO 2 catalysts. Through a series of carefully designed experiments, the pathway for the evolution of the Co cation-based molecular catalyst for the OER was further demonstrated on such a single Co-ion site for the first time. The Co 2+ ions were successively oxidized to Co 3+ and Co 4+ states prior to the OER. The sequential oxidation was coupled with the transfer of different numbers of protons/hydroxides and generated an active Co 4+ ═O fragment. A side-on hydroperoxo ligand of the Co 4+ site is proposed as a key intermediate for the formation of dioxygen.

  13. Unraveling the Nature of Active Sites in Ethanol Electro-oxidation by Site-Specific Marking of a Pt Catalyst with Isotope-Labeled 13CO.

    PubMed

    Farias, Manuel J S; Cheuquepán, William; Tanaka, Auro A; Feliu, Juan M

    2018-03-15

    This works deals with the identification of preferential site-specific activation at a model Pt surface during a multiproduct reaction. The (110)-type steps of a Pt(332) surface were selectively marked by attaching isotope-labeled 13 CO molecules to them, and ethanol oxidation was probed by in situ Foureir transfrom infrared spectroscopy in order to precisely determine the specific sites at which CO 2 , acetic acid, and acetaldehyde were preferentially formed. The (110) steps were active for splitting the C-C bond, but unexpectedly, we provide evidence that the pathway of CO 2 formation was preferentially activated at (111) terraces, rather than at (110) steps. Acetaldehyde was formed at (111) terraces at potentials comparable to those for CO 2 formation also at (111) terraces, while the acetic acid formation pathway became active only when the (110) steps were released by the oxidation of adsorbed 13 CO, at potentials higher than for the formation of CO 2 at (111) terraces of the stepped surface.

  14. AzaSite® inhibits Staphylococcus aureus and coagulase-negative Staphylococcus biofilm formation in vitro.

    PubMed

    Wu, Eric C; Kowalski, Regis P; Romanowski, Eric G; Mah, Francis S; Gordon, Y Jerold; Shanks, Robert M Q

    2010-12-01

    The aim of this study was to analyze the effect of azithromycin (AZM) 1% ophthalmic solution in DuraSite® (AzaSite®) on biofilm formation by Staphylococcus aureus and coagulase-negative staphylococci in vitro. Susceptible and resistant clinical strains (n = 8) of S. aureus and coagulase-negative staphylococci were challenged with serial dilutions of AzaSite® and its components: AZM, benzalkonium chloride (BAK), and the DuraSite drug delivery vehicle. After 20 h of incubation, bacterial growth was quantified using a spectrophotometer (A = 600 nm). Plates were stained with crystal violet and biofilm formation was quantified spectrophotometrically at A = 590 nm. AzaSite® and AZM inhibited bacterial growth (P < 0.05) and biofilm formation (P < 0.05) in AZM-susceptible strains at all studied dilutions. AZM-resistant strains treated with AzaSite® exhibited a significant reduction in biofilm formation (P < 0.05) at subinhibitory concentrations (1.25%-5%). AZM had no effect on bacterial growth in resistant strains but conferred a small reduction in biofilm formation at concentrations from 1.25 to 10 mg/mL in most strains. DuraSite® inhibited biofilm formation at concentrations between 10% and 2.5% in all studied strains (P < 0.05), without affecting bacterial growth. BAK inhibited bacterial growth and biofilm formation in all strains between concentrations of 0.042 and 0.375 mg/mL (P < 0.05). AzaSite®, AZM, or BAK prevented biofilm formation by inhibiting growth of AZM-susceptible strains. AzaSite®, AZM, and DuraSite® also reduced biofilm formation at subinhibitory concentrations for growth. Our data indicate that AZM has a moderate inhibitory effect on biofilm formation, whereas DuraSite® appears to play a greater role in the inhibition of staphylococcal biofilm formation by AzaSite®.

  15. Formative Evaluation of a Family Life Education Web Site

    ERIC Educational Resources Information Center

    Steimle, Brynn M.; Duncan, Stephen F.

    2004-01-01

    Hundreds of family life education Web sites are available on the Internet, allowing individuals and families unprecedented access to family life education information. Evaluation is critical to ensuring the quality of and improving these Web sites; yet, few Web site evaluations have been conducted. We formatively evaluated a new family life…

  16. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 -) to formate.

    PubMed

    Aslan, Aşkın Sevinç; Valjakka, Jarkko; Ruupunen, Jouni; Yildirim, Deniz; Turner, Nicholas J; Turunen, Ossi; Binay, Barış

    2017-01-01

    While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO 2 could also be utilized in the conversion of CO 2 to useful products via formate (HCOO - ). In this study, we investigated the reduction of CO 2 in the form of hydrogen carbonate (HCO 3 - ) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO 3 - as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO 3 - to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO 3 - reduced the reaction rate. CtFDH was modeled in the presence of HCO 3 - showing that it fits to the active site. The active site setting for hydride transfer in CO 2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO 3 - , resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

  18. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. © 2010 The Authors Journal compilation © 2010 FEBS.

  19. Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation.

    PubMed

    Xu, Binjie; Soukup, Randal J; Jones, Christopher J; Fishel, Richard; Wozniak, Daniel J

    2016-10-01

    During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely

  20. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

    PubMed

    Gerstmeier, Jana; Newcomer, Marcia E; Dennhardt, Sophie; Romp, Erik; Fischer, Jana; Werz, Oliver; Garscha, Ulrike

    2016-05-01

    Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. © FASEB.

  1. Nature of the Active Sites for CO Reduction on Copper Nanoparticles; Suggestions for Optimizing Performance.

    PubMed

    Cheng, Tao; Xiao, Hai; Goddard, William A

    2017-08-30

    Recent experiments show that the grain boundaries (GBs) of copper nanoparticles (NPs) lead to an outstanding performance in reducing CO 2 and CO to alcohol products. We report here multiscale simulations that simulate experimental synthesis conditions to predict the structure of a 10 nm Cu NP (158 555 atoms). To identify active sites, we first predict the CO binding at a large number of sites and select four exhibiting CO binding stronger than the (211) step surface. Then, we predict the formation energy of the *OCCOH intermediate as a descriptor for C-C coupling, identifying two active sites, both of which have an under-coordinated surface square site adjacent to a subsurface stacking fault. We then propose a periodic Cu surface (4 by 4 supercell) with a similar site that substantially decreases the formation energy of *OCCOH, by 0.14 eV.

  2. In situ formation of the active sites in Pd-Au bimetallic nanocatalysts for CO oxidation: NAP (near ambient pressure) XPS and MS study.

    PubMed

    Bukhtiyarov, A V; Prosvirin, I P; Saraev, A A; Klyushin, A Yu; Knop-Gericke, A; Bukhtiyarov, V I

    2018-06-07

    Model bimetallic Pd-Au/HOPG catalysts have been investigated in the CO oxidation reaction using a combination of NAP XPS and MS techniques. The samples have shown catalytic activity at temperatures above 150 °C. The redistribution of Au and Pd on the surface depending on the reaction conditions has been demonstrated using NAP XPS. The Pd enrichment of the bimetallic particles' surface under reaction gas mixture has been shown. Apparently, CO adsorption induces Pd segregation on the surface. Heating the sample under reaction conditions above 150 °C decomposes the Pd-CO state due to CO desorption and reaction and simultaneous Pd-Au alloy formation on the surface takes place. Cooling back down to RT results in reversible Pd segregation due to Pd-CO formation and the sample becomes inactive. It has been shown that in situ studies are necessary for investigation of the active sites in Pd-Au bimetallic systems.

  3. The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

    PubMed Central

    Just, Victoria J.; Burrell, Matthew R.; Bowater, Laura; McRobbie, Iain; Stevenson, Clare E. M.; Lawson, David M.; Bornemann, Stephen

    2007-01-01

    Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural. PMID:17680775

  4. View of Yellowknife Bay Formation, with Drilling Sites

    NASA Image and Video Library

    2013-12-09

    This mosaic of images from NASA Curiosity shows geological members of the Yellowknife Bay formation, and the sites where Curiosity drilled into the lowest-lying member, called Sheepbed, at targets John Klein and Cumberland.

  5. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  6. Water in the active site of ketosteroid isomerase.

    PubMed

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-08-09

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable

  7. Ultrafast Light-Driven Substrate Expulsion from the Active Site of a Photoswitchable Catalyst.

    PubMed

    Pescher, Manuel D; van Wilderen, Luuk J G W; Grützner, Susanne; Slavov, Chavdar; Wachtveitl, Josef; Hecht, Stefan; Bredenbeck, Jens

    2017-09-25

    The photoswitchable piperidine general base catalyst is a prototype structure for light control of catalysis. Its azobenzene moiety moves sterically shielding groups to either protect or expose the active site, thereby changing the basicity and hydrogen-bonding affinity of the compound. The reversible switching dynamics of the catalyst is probed in the infrared spectral range by monitoring hydrogen bond (HB) formation between its active site and methanol (MeOH) as HB donor. Steady-state infrared (IR) and ultrafast IR and UV/Vis spectroscopies are used to uncover ultrafast expulsion of MeOH from the active site within a few picoseconds. Thus, the force generated by the azobenzene moiety even in the final phase of its isomerization is sufficient to break a strong HB within 3 ps and to shut down access to the active site. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation.

    PubMed

    Friedt, Jenna; Leavens, Fern M V; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

    2014-04-01

    Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.

  9. An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

    PubMed Central

    Friedt, Jenna; Leavens, Fern M. V.; Mercier, Evan; Wieden, Hans-Joachim; Kothe, Ute

    2014-01-01

    Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism. PMID:24371284

  10. SERS-activating effect of chlorides on borate-stabilized silver nanoparticles: formation of new reduced adsorption sites and induced nanoparticle fusion.

    PubMed

    Sloufová, Ivana; Sisková, Karolína; Vlcková, Blanka; Stepánek, Josef

    2008-04-28

    Changes in morphology, surface reactivity and surface-enhancement of Raman scattering induced by modification of borate-stabilized Ag nanoparticles by adsorbed chlorides have been explored using TEM, EDX analysis and SERS spectra of probing adsorbate 2,2'-bipyridine (bpy) excited at 514.5 nm and evaluated by factor analysis. At fractional coverages of the parent Ag nanoparticles by adsorbed chlorides <0.6, the Ag colloid/Cl(-)/bpy systems were found to be constituted by fractal aggregates of Ag nanoparticles fairly uniform in size (10 +/- 2 nm) and SERS spectra of Ag(+)-bpy surface species were detected. The latter result was interpreted in terms of the presence of oxidized Ag(+) and/or Ag(n)(+) adsorption sites, which have been encountered also in systems with the chemically untreated Ag nanoparticles. At chloride coverages >0.6, a fusion of fractal aggregates into the compact aggregates of touching and/or interpenetrating Ag nanoparticles has been observed and found to be accompanied by the formation of another surface species, Ag-bpy, as well as by the increase of the overall SERS enhancement of bpy by factor of 40. The same Ag-bpy surface species has been detected under the strongly reducing conditions of reduction of silver nitrate by sodium borohydride in the presence of bpy. The formation of Ag-bpy is thus interpreted in terms of the stabilization of reduced Ag(0) adsorption sites by adsorbed bpy. The formation of reduced adsorption sites on Ag nanoparticle surfaces at chloride coverages >0.6 is discussed in terms of local changes in the work function of Ag. Finally, the SERS spectral detection of Ag-bpy species is proposed as a tool for probing the presence of reduced Ag(0) adsorption sites in systems with chemically modified Ag nanoparticles.

  11. Loss of Drosophila Vps16A enhances autophagosome formation through reduced Tor activity.

    PubMed

    Takáts, Szabolcs; Varga, Ágnes; Pircs, Karolina; Juhász, Gábor

    2015-01-01

    The HOPS tethering complex facilitates autophagosome-lysosome fusion by binding to Syx17 (Syntaxin 17), the autophagosomal SNARE. Here we show that loss of the core HOPS complex subunit Vps16A enhances autophagosome formation and slows down Drosophila development. Mechanistically, Tor kinase is less active in Vps16A mutants likely due to impaired endocytic and biosynthetic transport to the lysosome, a site of its activation. Tor reactivation by overexpression of Rheb suppresses autophagosome formation and restores growth and developmental timing in these animals. Thus, Vps16A reduces autophagosome numbers both by indirectly restricting their formation rate and by directly promoting their clearance. In contrast, the loss of Syx17 blocks autophagic flux without affecting the induction step in Drosophila.

  12. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Crichlow, G.; Lubetsky, J; Leng, L

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic datamore » indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.« less

  13. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    PubMed Central

    Wang, Yang-Gang; Mei, Donghai; Glezakou, Vassiliki-Alexandra; Li, Jun; Rousseau, Roger

    2015-01-01

    Catalysis by gold supported on reducible oxides has been extensively studied, yet issues such as the nature of the catalytic site and the role of the reducible support remain fiercely debated topics. Here we present ab initio molecular dynamics simulations of an unprecedented dynamic single-atom catalytic mechanism for the oxidation of carbon monoxide by ceria-supported gold clusters. The reported dynamic single-atom catalytic mechanism results from the ability of the gold cation to strongly couple with the redox properties of the ceria in a synergistic manner, thereby lowering the energy of redox reactions. The gold cation can break away from the gold nanoparticle to catalyse carbon monoxide oxidation, adjacent to the metal/oxide interface and subsequently reintegrate back into the nanoparticle after the reaction is completed. Our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in catalysis. PMID:25735407

  14. Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

    PubMed

    Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

    2017-07-21

    E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation.

    PubMed

    Ujike, Makoto; Nakajima, Katsuhisa; Nobusawa, Eri

    2004-11-01

    The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.

  16. Formation of marine snow and enhanced enzymatic activities in oil-contaminated seawater

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; McKay, L.; Yang, T.; Rhodes, B.; Nigro, L.; Gutierrez, T.; Teske, A.; Arnosti, C.

    2010-12-01

    The fate of oil spilled into the ocean depends on its composition, as well as on biological, chemical, and physical characteristics of the spill site. We investigated the effects of oil addition from the Deepwater Horizon (DH) spill on otherwise uncontaminated water collected close to the spill site. Incubation on a roller table mimicked the physical dynamics of natural seawater, leading to the formation of marine snow-oil aggregates. We measured the enzymatic activities of heterotrophic microbes associated with the aggregates and in the surrounding water, and assessed microbial population and community composition as oil-marine snow aggregates formed and aged in the water. Surface seawater taken near the spill site in May 2010 that had no visible crude oil was incubated in 1-l glass bottles with (oil-bottles) and without (no-oil bottles) a seawater-oil mixture collected from the same site. In the oil-bottles formation of brownish, densely packed marine snow (2-3 cm diameter) was observed within the first hour of the roller table incubation. In contrast no-oil bottles showed aggregate formation only after 3 days, and aggregates were almost transparent, less abundant, and smaller in size (< 1cm diameter). Subsamples of the water surrounding the aggregates were taken throughout 21 days of the roller table incubation, and analyzed for bacterial abundance and community structure as well as the activities of hydrolytic enzymes that are used by heterotrophic bacteria to degrade organic matter. We monitored oil-degrading activities with MUF-stearate and -butyrate, and also measured b-glucosidase, alkaline phosphatase, aminopeptidase, and six different polysaccharide hydrolase activities. Enzymatic activities were up to one order of magnitude higher in the oil-bottles compared with the no-oil bottles throughout the entire incubation time. Butyrate hydrolysis was elevated throughout the time course of the incubation, and stearate hydrolysis was particularly high over the

  17. Characterization of the active site properties of CYP4F12.

    PubMed

    Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

    2014-10-01

    Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Particle formation and growth at five rural and urban sites

    NASA Astrophysics Data System (ADS)

    Jeong, C.-H.; Evans, G. J.; McGuire, M. L.; Chang, R. Y.-W.; Abbatt, J. P. D.; Zeromskiene, K.; Mozurkewich, M.; Li, S.-M.; Leaitch, W. R.

    2010-08-01

    Ultrafine particle (UFP) number and size distributions were simultaneously measured at five urban and rural sites during the summer of 2007 in Ontario, Canada as part of the Border Air Quality and Meteorology Study (BAQS-Met 2007). Particle formation and growth events at these five sites were classified based on their strength and persistence as well as the variation in geometric mean diameter. Regional nucleation and growth events and local short-lived strong nucleation events were frequently observed at the near-border rural sites, upwind of industrial sources. Surprisingly, the particle number concentrations at one of these sites were higher than the concentrations at a downtown site in a major city, despite its high traffic density. Regional nucleation and growth events were favored during intense solar irradiance and in less polluted cooler drier air. The most distinctive regional particle nucleation and growth event during the campaign was observed simultaneously at all five sites, which were up to 350 km apart. Although the ultrafine particle concentrations and size distributions generally were spatially heterogeneous across the region, a more uniform spatial distribution of UFP across the five areas was observed during this regional nucleation event. Thus, nucleation events can cover large regions, contributing to the burden of UFP in cities and potentially to the associated health impacts on urban populations. Local short-lived nucleation events at the three near-border sites during this summer three-week campaign were associated with high SO2, which likely originated from US and Canadian industrial sources. Hence, particle formation in southwestern Ontario appears to often be related to anthropogenic gaseous emissions but biogenic emissions at times also contribute. Longer-term studies are needed to help resolve the relative contributions of anthropogenic and biogenic emissions to nucleation and growth in this region.

  19. Normal Modes Expose Active Sites in Enzymes.

    PubMed

    Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

    2016-12-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.

  20. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  1. Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent

    PubMed Central

    Itoh, Yoshiki; Cai, Kewen; Khorana, H. Gobind

    2001-01-01

    Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at λ > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the Tα subunit. Crosslinking to Tα was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in Tα were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19–Arg-28 at the N-terminal region of Tα. The total results show that both the N and the C termini of Tα are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T. PMID:11320238

  2. Active sites for CO 2 hydrogenation to methanol on Cu/ZnO catalysts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kattel, Shyam; Ramírez, Pedro J.; Chen, Jingguang G.

    The active sites over commercial copper/zinc oxide/aluminum oxide (Cu/ZnO/Al 2O 3) catalysts for carbon dioxide (CO 2) hydrogenation to methanol, the Zn-Cu bimetallic sites or ZnO-Cu interfacial sites, have recently been the subject of intense debate. Here, we report a direct comparison between the activity of ZnCu and ZnO/Cu model catalysts for methanol synthesis. By combining x-ray photoemission spectroscopy, density functional theory, and kinetic Monte Carlo simulations, we can identify and characterize the reactivity of each catalyst. Both experimental and theoretical results agree that ZnCu undergoes surface oxidation under the reaction conditions so that surface Zn transforms into ZnO andmore » allows ZnCu to reach the activity of ZnO/Cu with the same Zn coverage. These results highlight a synergy of Cu and ZnO at the interface that facilitates methanol synthesis via formate intermediates.« less

  3. Active sites for CO 2 hydrogenation to methanol on Cu/ZnO catalysts

    DOE PAGES

    Kattel, Shyam; Ramírez, Pedro J.; Chen, Jingguang G.; ...

    2017-03-23

    The active sites over commercial copper/zinc oxide/aluminum oxide (Cu/ZnO/Al 2O 3) catalysts for carbon dioxide (CO 2) hydrogenation to methanol, the Zn-Cu bimetallic sites or ZnO-Cu interfacial sites, have recently been the subject of intense debate. Here, we report a direct comparison between the activity of ZnCu and ZnO/Cu model catalysts for methanol synthesis. By combining x-ray photoemission spectroscopy, density functional theory, and kinetic Monte Carlo simulations, we can identify and characterize the reactivity of each catalyst. Both experimental and theoretical results agree that ZnCu undergoes surface oxidation under the reaction conditions so that surface Zn transforms into ZnO andmore » allows ZnCu to reach the activity of ZnO/Cu with the same Zn coverage. These results highlight a synergy of Cu and ZnO at the interface that facilitates methanol synthesis via formate intermediates.« less

  4. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

    PubMed Central

    Treuheit, Nicholas A.; Beach, Muneera A.; Komives, Elizabeth A.

    2011-01-01

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethylketone to the active site serine, as well as non-covalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1, however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-L-arginine-(3-methyl-1,5-pantanediyl) amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause the same reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or to exosite 1. PMID:21526769

  5. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

    PubMed

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

    2011-05-31

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  6. Three-dimensional representations of salt-dome margins at four active strategic petroleum reserve sites.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rautman, Christopher Arthur; Stein, Joshua S.

    2003-01-01

    Existing paper-based site characterization models of salt domes at the four active U.S. Strategic Petroleum Reserve sites have been converted to digital format and visualized using modern computer software. The four sites are the Bayou Choctaw dome in Iberville Parish, Louisiana; the Big Hill dome in Jefferson County, Texas; the Bryan Mound dome in Brazoria County, Texas; and the West Hackberry dome in Cameron Parish, Louisiana. A new modeling algorithm has been developed to overcome limitations of many standard geological modeling software packages in order to deal with structurally overhanging salt margins that are typical of many salt domes. Thismore » algorithm, and the implementing computer program, make use of the existing interpretive modeling conducted manually using professional geological judgement and presented in two dimensions in the original site characterization reports as structure contour maps on the top of salt. The algorithm makes use of concepts of finite-element meshes of general engineering usage. Although the specific implementation of the algorithm described in this report and the resulting output files are tailored to the modeling and visualization software used to construct the figures contained herein, the algorithm itself is generic and other implementations and output formats are possible. The graphical visualizations of the salt domes at the four Strategic Petroleum Reserve sites are believed to be major improvements over the previously available two-dimensional representations of the domes via conventional geologic drawings (cross sections and contour maps). Additionally, the numerical mesh files produced by this modeling activity are available for import into and display by other software routines. The mesh data are not explicitly tabulated in this report; however an electronic version in simple ASCII format is included on a PC-based compact disk.« less

  7. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    PubMed

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

  8. A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity.

    PubMed

    Tilvawala, Ronak; Cammarata, Michael; Adediran, S A; Brodbelt, Jennifer S; Pratt, R F

    2015-12-22

    O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

  9. The Strength of an Ig Switch Region is Determined by its Ability to Drive R-loop Formation and its Number of WGCW Sites

    PubMed Central

    Zhang, Zheng Z.; Pannunzio, Nicholas R.; Han, Li; Hsieh, Chih-Lin; Yu, Kefei; Lieber, Michael R.

    2014-01-01

    SUMMARY R-loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R-loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR, regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R-loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R-loop formation, and on the number of WGCW sites. PMID:25017067

  10. Directing reaction pathways by catalyst active-site selection using self-assembled monolayers.

    PubMed

    Pang, Simon H; Schoenbaum, Carolyn A; Schwartz, Daniel K; Medlin, J Will

    2013-01-01

    One key route for controlling reaction selectivity in heterogeneous catalysis is to prepare catalysts that exhibit only specific types of sites required for desired product formation. Here we show that alkanethiolate self-assembled monolayers with varying surface densities can be used to tune selectivity to desired hydrogenation and hydrodeoxygenation products during the reaction of furfural on supported palladium catalysts. Vibrational spectroscopic studies demonstrate that the selectivity improvement is achieved by controlling the availability of specific sites for the hydrogenation of furfural on supported palladium catalysts through the selection of an appropriate alkanethiolate. Increasing self-assembled monolayer density by controlling the steric bulk of the organic tail ligand restricts adsorption on terrace sites and dramatically increases selectivity to desired products furfuryl alcohol and methylfuran. This technique of active-site selection simultaneously serves both to enhance selectivity and provide insight into the reaction mechanism.

  11. Acute tissue injury activates satellite cells and promotes sarcoma formation via the HGF/c-MET signaling pathway.

    PubMed

    Van Mater, David; Añó, Leonor; Blum, Jordan M; Webster, Micah T; Huang, WeiQiao; Williams, Nerissa; Ma, Yan; Cardona, Diana M; Fan, Chen-Ming; Kirsch, David G

    2015-02-01

    Some patients with soft-tissue sarcoma (STS) report a history of injury at the site of their tumor. Although this phenomenon is widely reported, there are relatively few experimental systems that have directly assessed the role of injury in sarcoma formation. We recently described a mouse model of STS whereby p53 is deleted and oncogenic Kras is activated in muscle satellite cells via a Pax7(CreER) driver following intraperitoneal injection with tamoxifen. Here, we report that after systemic injection of tamoxifen, the vast majority of Pax7-expressing cells remain quiescent despite mutation of p53 and Kras. The fate of these muscle progenitors is dramatically altered by tissue injury, which leads to faster kinetics of sarcoma formation. In adult muscle, quiescent satellite cells will transition into an active state in response to hepatocyte growth factor (HGF). We show that modulating satellite cell quiescence via intramuscular injection of HGF increases the penetrance of sarcoma formation at the site of injection, which is dependent on its cognate receptor c-MET. Unexpectedly, the tumor-promoting effect of tissue injury also requires c-Met. These results reveal a mechanism by which HGF/c-MET signaling promotes tumor formation after tissue injury in a mouse model of primary STS, and they may explain why some patients develop a STS at the site of injury. ©2014 American Association for Cancer Research.

  12. The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

    PubMed

    Skov, Lars K; Pizzut-Serin, Sandra; Remaud-Simeon, Magali; Ernst, Heidi A; Gajhede, Michael; Mirza, Osman

    2013-09-01

    Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

  13. Authigenic Carbonate Formation on the Peru Margin; New Insights from IODP Site 1230

    NASA Astrophysics Data System (ADS)

    Abdullajintakam, S.; Naehr, T. H.

    2015-12-01

    Fluid seepage of reduced organic compounds such as methane impacts the geology and biology of the seabed by inducing complex, microbially mediated biogeochemical processes. Authigenic carbonates serve as one of the few permanent records of these of dynamic biogeochemical interactions that involve methanogenesis, methanotrophy, sulfate reduction and carbonate precipitation. Meister et al. (2007) investigated deep-sea dolomite formation at Sites 1227-1229 on the Peru margin, where dolomite precipitation occurs in association with organic carbon-rich continental margin sediments. Geochemical and petrographic studies indicated episodic dolomite precipitation at a dynamic sulfate methane transition zone (SMTZ). Variations in δ13C values of these dolomites between +15‰ and -15‰ were attributed to non-steady state conditions as a result of the upward and downward migration of the SMTZ. Our study aims to better understand the biogeochemical processes associated with authigenic carbonate precipitation in this dynamic deep-sea setting. We focused our efforts on IODP Site 1230, which is a gas-hydrate-bearing site that shows sulphate consumption within the uppermost 10 m below the seafloor as well as high methane production. Using a multi proxy approach, we combined X-ray diffraction, stable isotope geochemistry, and trace metal analysis of authigenic carbonates to elucidate conditions for authigenic carbonate formation. Results from Site 1230 are compared to Sites 1227 and 1229, which lacks gas hydrates and is characterized by high pore water sulfate and low methane concentrations. This study contributes to a more comprehensive understanding of authigenic carbonate formation and associated biogeochemical processes in continental margin sediments. Meister, P., Mckenzie, J. A., Vasconcelos, C., Bernasconi, S., Frank, M., Gutjhar, M. and SCHRAG, D. P. (2007), Dolomite formation in the dynamic deep biosphere: results from the Peru Margin. Sedimentology, 54: 1007-1032.

  14. Ground-water flow model of the Boone formation at the Tar Creek superfund site, Oklahoma and Kansas

    USGS Publications Warehouse

    Reed, T.B.; Czarnecki, John B.

    2006-01-01

    Extensive mining activities conducted at the Tar Creek Superfund site, one of the largest Superfund sites in the United States, pose substantial health and safety risks. Mining activities removed a total of about 6,000,000 tons of lead and zinc by 1949. To evaluate the effect of this mining on the ground-water flow, a MODFLOW 2000 digital model has been developed to simulate ground-water flow in the carbonate formations of Mississippian age underlying the Tar Creek Superfund site. The model consists of three layers of variable thickness and a grid of 580 rows by 680 columns of cells 164 feet (50 meters) on a side. Model flux boundary conditions are specified for rivers and general head boundaries along the northern boundary of the Boone Formation. Selected cells in layer 1 are simulated as drain cells. Model calibration has been performed to minimize the difference between simulated and observed water levels in the Boone Formation. Hydraulic conductivity values specified during calibration range from 1.3 to 35 feet per day for the Boone Formation with the larger values occurring along the axis of the Miami Syncline where horizontal anisotropy is specified as 10 to 1. Hydraulic conductivity associated with the mine void is set at 50,000 feet per day and a specific yield of 1.0 is specified to represent that the mine void is filled completely with water. Residuals (the difference between measured and simulated ground-water altitudes) has a root-mean-squared value of 8.53 feet and an absolute mean value of 7.29 feet for 17 observed values of water levels in the Boone Formation. The utility of the model for simulating and evaluating the possible consequences of remediation activities has been demonstrated. The model was used to simulate the emplacement of chat (mine waste consisting of fines and fragments of chert) back into the mine. Scenarios using 1,800,000 and 6,500,000 tons of chat were run. Hydraulic conductivity was reduced from 50,000 feet per day to 35 feet

  15. Post-formation copper-nitrogen species on carbon black: their chemical structures and active sites for oxygen reduction reaction.

    PubMed

    Xie, Xin; Liu, Jingjun; Li, Tuanfeng; Song, Ye; Wang, Feng

    2018-05-16

    Note that 3d transition metal and nitrogen co-doped carbon materials (TM-N-C) are considered as the most promising next-generation electrocatalysts alternative to precious Pt for oxygen reduction reaction (ORR). Herein, we have fabricated a Cu-N-C catalyst through directly grafting copper-nitrogen complexes composed by cuprous chloride and ammonia water onto the surface of an industrial carbon black at 500℃. In an alkaline environment, the synthesized catalyst exhibits excellent ORR catalytic activity, which is comparable to the state-of-the-art Pt/C catalyst but far exceeding that obtained by the original carbon. Moreover, the catalyst displays much better stability than the Pt/C. The enhanced ORR performance is proven to originate from the post-formation Cu(I)-N2 and Cu(II)-N4 sites at the carbon surface, as evidenced by X-ray photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (XAS). The possible ORR process catalyzed by these Cu-Nx species is discussed at atomic level. This work provides a simple and fast synthesis strategy for efficient TM-N-C catalysts on a large scale for energy storage and conversion systems. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Delayed condensation and frost formation on superhydrophobic carbon soot coatings by controlling the presence of hydrophilic active sites

    NASA Astrophysics Data System (ADS)

    Esmeryan, Karekin D.; Castano, Carlos E.; Mohammadi, Reza; Lazarov, Yuliyan; Radeva, Ekaterina I.

    2018-02-01

    Condensation frosting is an undesired natural phenomenon that could be impeded efficiently using appropriate wettability and morphologically patterned surfaces. The icephobic properties of carbon soot and the fabrication scalability of its synthesis method are a good foundation for anti-frosting applications; however, the fundamentals of frost growth and spreading on sooted surfaces have not been examined yet. In this study, we investigate the anti-frosting performance of three groups of superhydrophobic soot coatings by means of 16 MHz quartz crystal microbalances (QCMs). The analysis of the real-time sensor signal of each soot coated QCM pattern shows that frost formation and its propagation velocity depend on the quantity of oxygen functionalities and structural defects in the material. In turn, the reduction of both parameters shifts the onset of frost growth to temperatures below  -20 °C, whereas the interdroplet ice bridging is slowed by a factor of four. Moreover, high-resolution scanning electron micrographs of the samples imply delamination upon defrosting of the soot with spherical-like morphology via polar interactions driven mechanism. These results reveal an opportunity for control of frost incipiency on sooted surfaces by adjusting the synthesis conditions and depositing soot coatings with as low as possible content of hydrophilic active sites.

  17. An evaluation of water quality in private drinking water wells near natural gas extraction sites in the Barnett Shale formation.

    PubMed

    Fontenot, Brian E; Hunt, Laura R; Hildenbrand, Zacariah L; Carlton, Doug D; Oka, Hyppolite; Walton, Jayme L; Hopkins, Dan; Osorio, Alexandra; Bjorndal, Bryan; Hu, Qinhong H; Schug, Kevin A

    2013-09-03

    Natural gas has become a leading source of alternative energy with the advent of techniques to economically extract gas reserves from deep shale formations. Here, we present an assessment of private well water quality in aquifers overlying the Barnett Shale formation of North Texas. We evaluated samples from 100 private drinking water wells using analytical chemistry techniques. Analyses revealed that arsenic, selenium, strontium and total dissolved solids (TDS) exceeded the Environmental Protection Agency's Drinking Water Maximum Contaminant Limit (MCL) in some samples from private water wells located within 3 km of active natural gas wells. Lower levels of arsenic, selenium, strontium, and barium were detected at reference sites outside the Barnett Shale region as well as sites within the Barnett Shale region located more than 3 km from active natural gas wells. Methanol and ethanol were also detected in 29% of samples. Samples exceeding MCL levels were randomly distributed within areas of active natural gas extraction, and the spatial patterns in our data suggest that elevated constituent levels could be due to a variety of factors including mobilization of natural constituents, hydrogeochemical changes from lowering of the water table, or industrial accidents such as faulty gas well casings.

  18. Stabilization and activation of alpha-chymotrypsin in water-organic solvent systems by complex formation with oligoamines.

    PubMed

    Kudryashova, Elena V; Artemova, Tatiana M; Vinogradov, Alexei A; Gladilin, Alexander K; Mozhaev, Vadim V; Levashov, Andrey V

    2003-04-01

    Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.

  19. Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

    PubMed

    Adamczyk, Katrin; Simpson, Niall; Greetham, Gregory M; Gumiero, Andrea; Walsh, Martin A; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

    2015-01-01

    Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H 2 O and D 2 O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump-probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.

  20. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

    PubMed Central

    Sengupta, Raghuvir N.; Van Schie, Sabine N.S.; Giambaşu, George; Dai, Qing; Yesselman, Joseph D.; York, Darrin; Piccirilli, Joseph A.; Herschlag, Daniel

    2016-01-01

    Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

  1. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

    PubMed

    Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

    2016-01-01

    Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. © 2015 Sengupta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  2. Simultaneous measurements of new particle formation at 1 s time resolution at a street site and a rooftop site

    NASA Astrophysics Data System (ADS)

    Zhu, Yujiao; Yan, Caiqing; Zhang, Renyi; Wang, Zifa; Zheng, Mei; Gao, Huiwang; Gao, Yang; Yao, Xiaohong

    2017-08-01

    This study is the first to use two identical Fast Mobility Particle Sizers for simultaneous measurement of particle number size distributions (PNSDs) at a street site and a rooftop site within 500 m distance in wintertime and springtime to investigate new particle formation (NPF) in Beijing. The collected datasets at 1 s time resolution allow deduction of the freshly emitted traffic particle signal from the measurements at the street site and thereby enable the evaluation of the effects on NPF in an urban atmosphere through a site-by-site comparison. The number concentrations of 8 to 20 nm newly formed particles and the apparent formation rate (FR) in the springtime were smaller at the street site than at the rooftop site. In contrast, NPF was enhanced in the wintertime at the street site with FR increased by a factor of 3 to 5, characterized by a shorter NPF time and higher new particle yields than at the rooftop site. Our results imply that the street canyon likely exerts distinct effects on NPF under warm or cold ambient temperature conditions because of on-road vehicle emissions, i.e., stronger condensation sinks that may be responsible for the reduced NPF in the springtime but efficient nucleation and partitioning of gaseous species that contribute to the enhanced NPF in the wintertime. The occurrence or absence of apparent growth for new particles with mobility diameters larger than 10 nm was also analyzed. The oxidization of biogenic organics in the presence of strong photochemical reactions is suggested to play an important role in growing new particles with diameters larger than 10 nm, but sulfuric acid is unlikely to be the main species for the apparent growth. However, the number of datasets used in this study is relatively small, and larger datasets are essential to draw a general conclusion.

  3. DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

    NASA Astrophysics Data System (ADS)

    Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

    2011-07-01

    DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

  4. Mineral abundances at the final four curiosity study sites and implications for their formation

    NASA Astrophysics Data System (ADS)

    Poulet, F.; Carter, J.; Bishop, J. L.; Loizeau, D.; Murchie, S. M.

    2014-03-01

    A component of the landing site selection process for the Mars Science Laboratory (MSL) involved the presence of phyllosilicates as the main astrobiological targets. Gale crater was selected as the MSL landing site from among 4 down selected study sites (Gale, Eberswalde and Holden craters, Mawrth Vallis) that addressed the primary scientific goal of assessing the past habitability of Mars. A key constraint on the formation process of these phyllosilicate-bearing deposits is in the precise mineralogical composition. We present a reassessment of the mineralogy of the sites combined with a determination of the modal mineralogy of the major phyllosilicate-bearing deposits of the four final study sites from the modeling of near-infrared spectra using a radiative transfer model. The largest abundance of phyllosilicates (30-70%) is found in Mawrth Vallis, the lowest one in Eberswalde (<25%). Except for Mawrth Vallis, the anhydrous phases (plagioclase, pyroxenes and martian dust) are the dominant phases, suggesting formation conditions with a lower alteration grade and/or a post-formation mixing with anhydrous phases. The composition of Holden layered deposits (mixture of saponite and micas with a total abundance in the range of 25-45%) suggests transport and deposition of altered basalts of the Noachian crust without major chemical transformation. For Eberswalde, the modal mineralogy is also consistent with detrital clays, but the presence of opaline silica indicates that an authigenic formation occurred during the deposition. The overall composition including approximately 20-30% smectite detected by MSL in the rocks of Yellow-knife Bay area interpreted to be material deposited on the floor of Gale crater by channels (http://www.nasa.gov/mission_pages/msl/news/msl20130312.html).

  5. Influences of aerosol physiochemical properties and new particle formation on CCN activity from observation at a suburban site of China

    NASA Astrophysics Data System (ADS)

    Li, Yanan; Zhang, Fang; Li, Zhanqing; Sun, Li; Wang, Zhenzhu; Li, Ping; Sun, Yele; Ren, Jingye; Wang, Yuying; Cribb, Maureen; Yuan, Cheng

    2017-05-01

    With the aim of understanding the impact of aerosol particle size and chemical composition on CCN activity, the size-resolved cloud condensation nuclei (CCN) number concentration (NCCN), particle number size distribution (PSD) (10-600 nm), and bulk chemical composition of particles with a diameter < 1.0 μm (PM1) were measured simultaneously at Xinzhou, a suburban site in northern China, from 22 July to 26 August 2014. The NCCN was measured at five different supersaturations (SS) ranging from 0.075%-0.76%. Diurnal variations in the aerosol number concentration (NCN), NCCN, the bulk aerosol activation ratio (AR), the hygroscopicity parameter (κchem), and the ratio of 44 mass to charge ration (m/z 44) to total organic signal in the component spectrum (f44), and the PSD were examined integrally to study the influence of particle size and chemical composition on CCN activation. We found that particle size was more related to the CCN activation ratios in the morning, whereas in the afternoon ( 1400 LST), κchem and f44 were more closely associated with the bulk AR. Assuming the internal mixing of aerosol particles, NCCN was estimated using the bulk chemical composition and real-time PSD. We found that the predicted CCN number concentrations were underestimated by 20-30% at SS < 0.2% probably due to the measurement uncertainties. Estimates were more accurate at higher SS levels, suggesting that the hygroscopicity parameter based on bulk chemical composition information can provide a good estimate of CCN number concentrations. We studied the impacts of new particle formation (NPF) events on size-resolved CCN activity at the ;growth; stage and ;leveling-off; stage during a typical NPF event by comparing with the case during non-NPF event. It has been found that CCN activation was restrained at the ;growth; stage during which larger particle diameters were needed to reach an activation diameter(Da), and the bulk AR decreased as well. However, during the ;leveling

  6. Multiscalar approach to archaeological site formation at GaJj17, East Turkana, Kenya

    NASA Astrophysics Data System (ADS)

    Murray, B. M.; Ranhorn, K. L.; Colarossi, D.; Mavuso, S. S.; Dogandžić, T.; Ziegler, M. J.; Warren, S. L.; Braun, D. R.; Harris, J. W. K.

    2017-12-01

    Kenya's East Turkana region hosts a rich Plio­Pleistocene record of fossils, archaeological artifacts, and sedimentary features whose chronostratigraphic histories are often obscured by landscape changes from erosional events and tectonic activity. The Middle Stone Age (MSA) record of the Koobi Fora Formation (KF Fm.) has particularly been subjected to this complex depositional history, making it a sparse unit and, consequently, widely understudied. Stratigraphically located in between the maximum capping unconformity of the KF Fm.'s Chari tuff ( 1.39 Ma) and that of the Galana Boi Fm. ( 10 ka), the unit provides a unique window into understanding the Late Pleistocene of the region. The MSA surface scatters at archaeological site GaJj17 prompted further study into the site's age and depositional chronology. The GaJj17 ridge is locally distinguished by its cap of Late Pleistocene sands overlying strata containing tuffs likely of the Upper Burgi (2.0­-1.87 Ma) or KBS (1.87­-1.56 Ma) members. To investigate whether GaJj17's preservation is due to tectonic deformation, a broader scale examination of the structural geology was conducted through surveys and aerial imagery. Regions of deformation were identified and mapped to establish the geological history of the locality. Resultant observations and elevation data offer insight into regional faults at the root of prolonged structural alterations which have facilitated the unique preservation of MSA materials. Through a multiscalar approach it is possible to understand both the formation of GaJj17 and the underlying processes behind preservation and destruction in the changing landscape of the Turkana basin, enabling future identification of archaeological sites through proxies of elevation, regional stratigraphy, and fault mapping. This research was supported by IRES grants 1358178 and 1358200 from the U.S. National Science Foundation.

  7. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  8. Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

    PubMed

    Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

    2018-02-02

    Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Slow Starter Enzymes: Role of Active Site Architecture in the Catalytic Control in the Biosynthesis of Taxadiene by Taxadiene Synthase.

    PubMed

    Ansbacher, Tamar; Freud, Yehoshua; Major, Dan Thomas

    2018-05-23

    Taxadiene synthase (TXS) catalyzes the formation of the natural product Taxa-4(5),11(12)-diene (henceforth Taxadiene). Taxadiene is the precursor in the formation of Taxol, which is an important natural anti-cancer agent. In the current study, we present a detailed mechanistic view of the biosynthesis of Taxadiene by TXS, using a hybrid quantum mechanics-molecular mechanics potential in conjunction with free energy simulation methods. The obtained free energy landscape displays initial endergonic steps followed by a step-wise downhill profile, which is an emerging free energy fingerprint for type I terpene synthases. We identify an active site Trp residue (W753) as a key feature of the TXS active site architecture and propose that this residue stabilized intermediate cations via -cation interactions. To validate our proposed active TXS model, we examine a previously reported W753H mutation, which leads to exclusive formation of the side product, cembrene A. The simulations of the W753H mutant show that in the mutant structure, the His side-chain is in perfect position to deprotonate the cembrenyl cation en route to cembrene formation, and that this abortive deprotonation is an energetically facile process. Based on the current model, we propose that an analogous mutation of Y841 to His could possibly lead to verticillane. The current simulations stress the importance of precise positioning of key active site residues in stabilizing intermediate carbocations. In view of the great pharmaceutical importance of taxadiene, a detailed understanding of the TXS mechanism can provide important clues towards a synthetic strategy for taxol manufacturing.

  10. Active site dynamics of ribonuclease.

    PubMed Central

    Brünger, A T; Brooks, C L; Karplus, M

    1985-01-01

    The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state. Images PMID:3866234

  11. p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding.

    PubMed

    Itakura, Eisuke; Mizushima, Noboru

    2011-01-10

    Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

  12. BOREAS TE-20 Soils Data Over the NSA-MSA and Tower Sites in Raster Format

    NASA Technical Reports Server (NTRS)

    Hall, Forrest G. (Editor); Veldhuis, Hugo; Knapp, David; Veldhuis, Hugo

    2000-01-01

    The BOREAS TE-20 team collected several data sets for use in developing and testing models of forest ecosystem dynamics. This data set was gridded from vector layers of soil maps that were received from Dr. Hugo Veldhuis, who did the original mapping in the field during 1994. The vector layers were gridded into raster files that cover the NSA-MSA and tower sites. The data are stored in binary, image format files. The data files are available on a CD-ROM (see document number 20010000884), or from the Oak Ridge National Laboratory (ORNL) Distributed Active Center (DAAC).

  13. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  14. Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3.

    PubMed

    Shen, Hujun; Deng, Mingsen; Zhang, Yachao

    2017-10-01

    Recent crystal structures of RNA-dependent RNA polymerase (3D pol ) from Coxsackievirus B3 (CVB3) revealed that a tyrosine mutation at Phe364 (F364Y) resulted in structures with open active site whereas a hydrophobic mutation at Phe364 (F364A) led to conformations with closed active site. Besides, the crystal structures showed that the F364W mutation had no preference between the open and closed active sites, similar to wild-type. In this paper, we present a molecular dynamics (MD) study on CVB3 3D pol in order to address some important questions raised by experiments. First, MD simulations of F364Y and F364A were carried out to explore how these mutations at Phe364 influence active site dynamics and conformations. Second, MD simulations of wild-type and mutants were performed to discover the connection between active site dynamics and polymerase function. MD simulations reveal that the effect of mutations on active site dynamics is associated with the interaction between the structural motifs A and D in CVB3 3D pol . Interestingly, we discover that the active site state is influenced by the formation of a hydrogen bond between backbone atoms of Ala231 (in motif A) and Ala358 (in motif D), which has never been revealed before. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  16. Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

    PubMed Central

    Cai, Kewen; Itoh, Yoshiki; Khorana, H. Gobind

    2001-01-01

    Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345. PMID:11320237

  17. Site specific measurements of bone formation using [18F] sodium fluoride PET/CT

    PubMed Central

    Puri, Tanuj; Siddique, Musib; Frost, Michelle L.; Moore, Amelia E. B.; Fogelman, Ignac

    2018-01-01

    Dynamic positron emission tomography (PET) imaging with fluorine-18 labelled sodium fluoride ([18F]NaF) allows the quantitative assessment of regional bone formation by measuring the plasma clearance of fluoride to bone at any site in the skeleton. Today, hybrid PET and computed tomography (CT) dual-modality systems (PET/CT) are widely available, and [18F]NaF PET/CT offers a convenient non-invasive method of studying bone formation at the important osteoporotic fracture sites at the hip and spine, as well as sites of pure cortical or trabecular bone. The technique complements conventional measurements of bone turnover using biochemical markers or bone biopsy as a tool to investigate new therapies for osteoporosis, and has a potential role as an early biomarker of treatment efficacy in clinical trials. This article reviews methods of acquiring and analyzing dynamic [18F]NaF PET/CT scan data, and outlines a simplified approach combining venous blood sampling with a series of short (3- to 5-minute) static PET/CT scans acquired at different bed positions to estimate [18F]NaF plasma clearance at multiple sites in the skeleton with just a single injection of tracer. PMID:29541623

  18. Site specific measurements of bone formation using [18F] sodium fluoride PET/CT.

    PubMed

    Blake, Glen M; Puri, Tanuj; Siddique, Musib; Frost, Michelle L; Moore, Amelia E B; Fogelman, Ignac

    2018-02-01

    Dynamic positron emission tomography (PET) imaging with fluorine-18 labelled sodium fluoride ([ 18 F]NaF) allows the quantitative assessment of regional bone formation by measuring the plasma clearance of fluoride to bone at any site in the skeleton. Today, hybrid PET and computed tomography (CT) dual-modality systems (PET/CT) are widely available, and [ 18 F]NaF PET/CT offers a convenient non-invasive method of studying bone formation at the important osteoporotic fracture sites at the hip and spine, as well as sites of pure cortical or trabecular bone. The technique complements conventional measurements of bone turnover using biochemical markers or bone biopsy as a tool to investigate new therapies for osteoporosis, and has a potential role as an early biomarker of treatment efficacy in clinical trials. This article reviews methods of acquiring and analyzing dynamic [ 18 F]NaF PET/CT scan data, and outlines a simplified approach combining venous blood sampling with a series of short (3- to 5-minute) static PET/CT scans acquired at different bed positions to estimate [ 18 F]NaF plasma clearance at multiple sites in the skeleton with just a single injection of tracer.

  19. Active site architecture of a sugar N-oxygenase.

    PubMed

    Thoden, James B; Branch, Megan C; Zimmer, Alex L; Bruender, Nathan A; Holden, Hazel M

    2013-05-14

    KijD3 is a flavin-dependent N-oxygenase implicated in the formation of the nitro-containing sugar d-kijanose, found attached to the antibiotic kijanimicin. For this investigation, the structure of KijD3 in complex with FMN and its dTDP-sugar substrate was solved to 2.1 Å resolution. In contrast to the apoenzyme structure, the C-terminus of the protein becomes ordered and projects into the active site cleft [Bruender, N. A., Thoden, J. B., and Holden, H. M. (2010) Biochemistry 49, 3517-3524]. The amino group of the dTDP-aminosugar that is oxidized is located 4.9 Å from C4a of the flavin ring. The model provides a molecular basis for understanding the manner in which KijD3 catalyzes its unusual chemical transformation.

  20. Assessment of the potential for karst in the Rustler Formation at the WIPP site.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lorenz, John Clay

    2006-01-01

    This report is an independent assessment of the potential for karst dissolution in evaporitic strata of the Rustler Formation at the Waste Isolation Pilot Plant (WIPP) site. Review of the available data suggests that the Rustler strata thicken and thin across the area in depositional patterns related to lateral variations in sedimentary accommodation space and normal facies changes. Most of the evidence that has been offered for the presence of karst in the subsurface has been used out of context, and the different pieces are not mutually supporting. Outside of Nash Draw, definitive evidence for the development of karst inmore » the Rustler Formation near the WIPP site is limited to the horizon of the Magenta Member in drillhole WIPP-33. Most of the other evidence cited by the proponents of karst is more easily interpreted as primary sedimentary structures and the localized dissolution of evaporitic strata adjacent to the Magenta and Culebra water-bearing units. Some of the cited evidence is invalid, an inherited baggage from studies made prior to the widespread knowledge of modern evaporite depositional environments and prior to the existence of definitive exposures of the Rustler Formation in the WIPP shafts. Some of the evidence is spurious, has been taken out of context, or is misquoted. Lateral lithologic variations from halite to mudstone within the Rustler Formation under the WIPP site have been taken as evidence for the dissolution of halite such as that seen in Nash Draw, but are more rationally explained as sedimentary facies changes. Extrapolation of the known karst features in Nash Draw eastward to the WIPP site, where conditions are and have been significantly different for half a million years, is unwarranted. The volumes of insoluble material that would remain after dissolution of halite would be significantly less than the observed bed thicknesses, thus dissolution is an unlikely explanation for the lateral variations from halite to mudstone and

  1. A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

    PubMed

    Siddique, Shahid; Radakovic, Zoran S; De La Torre, Carola M; Chronis, Demosthenis; Novák, Ondřej; Ramireddy, Eswarayya; Holbein, Julia; Matera, Christiane; Hütten, Marion; Gutbrod, Philipp; Anjam, Muhammad Shahzad; Rozanska, Elzbieta; Habash, Samer; Elashry, Abdelnaser; Sobczak, Miroslaw; Kakimoto, Tatsuo; Strnad, Miroslav; Schmülling, Thomas; Mitchum, Melissa G; Grundler, Florian M W

    2015-10-13

    Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

  2. p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

    PubMed Central

    Itakura, Eisuke

    2011-01-01

    Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway. PMID:21220506

  3. Site Formation Processes and Hunter-Gatherers Use of Space in a Tropical Environment: A Geo-Ethnoarchaeological Approach from South India

    PubMed Central

    Friesem, David E.; Lavi, Noa; Madella, Marco; Ajithprasad, P.; French, Charles

    2016-01-01

    Hunter-gatherer societies have distinct social perceptions and practices which are expressed in unique use of space and material deposition patterns. However, the identification of archaeological evidence associated with hunter-gatherer activity is often challenging, especially in tropical environments such as rainforests. We present an integrated study combining ethnoarchaeology and geoarchaeology in order to study archaeological site formation processes related to hunter-gatherers’ ways of living in tropical forests. Ethnographic data was collected from an habitation site of contemporary hunter-gatherers in the forests of South India, aimed at studying how everyday activities and way of living dictate patterns of material deposition. Ethnoarchaeological excavations of abandoned open-air sites and a rock-shelter of the same group located deep in the forests, involved field observations and sampling of sediments from the abandoned sites and the contemporary site. Laboratory analyses included geochemical analysis (i.e., FTIR, ICP-AES), phytolith concentration analysis and soil micromorphology. The results present a dynamic spatial deposition pattern of macroscopic, microscopic and chemical materials, which stem from the distinctive ways of living and use of space by hunter-gatherers. This study shows that post-depositional processes in tropical forests result in poor preservation of archaeological materials due to acidic conditions and intensive biological activity within the sediments. Yet, the multiple laboratory-based analyses were able to trace evidence for activity surfaces and their maintenance practices as well as localized concentrations of activity remains such as the use of plants, metals, hearths and construction materials. PMID:27783683

  4. The Diverse AAA+ Machines that Repair Inhibited Rubisco Active Sites

    PubMed Central

    Mueller-Cajar, Oliver

    2017-01-01

    Gaseous carbon dioxide enters the biosphere almost exclusively via the active site of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This highly conserved catalyst has an almost universal propensity to non-productively interact with its substrate ribulose 1,5-bisphosphate, leading to the formation of dead-end inhibited complexes. In diverse autotrophic organisms this tendency has been counteracted by the recruitment of dedicated AAA+ (ATPases associated with various cellular activities) proteins that all use the energy of ATP hydrolysis to remodel inhibited Rubisco active sites leading to release of the inhibitor. Three evolutionarily distinct classes of these Rubisco activases (Rcas) have been discovered so far. Green and red-type Rca are mostly found in photosynthetic eukaryotes of the green and red plastid lineage respectively, whereas CbbQO is associated with chemoautotrophic bacteria. Ongoing mechanistic studies are elucidating how the various motors are utilizing both similar and contrasting strategies to ultimately perform their common function of cracking the inhibited Rubisco active site. The best studied mechanism utilized by red-type Rca appears to involve transient threading of the Rubisco large subunit C-terminal peptide, reminiscent of the action performed by Clp proteases. As well as providing a fascinating example of convergent molecular evolution, Rca proteins can be considered promising crop-improvement targets. Approaches aiming to replace Rubisco in plants with improved enzymes will need to ensure the presence of a compatible Rca protein. The thermolability of the Rca protein found in crop plants provides an opportunity to fortify photosynthesis against high temperature stress. Photosynthesis also appears to be limited by Rca when light conditions are fluctuating. Synthetic biology strategies aiming to enhance the autotrophic CO2 fixation machinery will need to take into consideration the requirement for Rubisco activases

  5. O–O bond formation in ruthenium-catalyzed water oxidation: single-site nucleophilic attack vs. O–O radical coupling

    DOE PAGES

    Shaffer, David W.; Xie, Yan; Concepcion, Javier J.

    2017-09-01

    In this review we discuss at the mechanistic level the different steps involved in water oxidation catalysis with ruthenium-based molecular catalysts. We have chosen to focus on ruthenium-based catalysts to provide a more coherent discussion and because of the availability of detailed mechanistic studies for these systems but many of the aspects presented in this review are applicable to other systems as well. The water oxidation cycle has been divided in four major steps: water oxidative activation, O–O bond formation, oxidative activation of peroxide intermediates, and O 2 evolution. A significant portion of the review is dedicated to the O–Omore » bond formation step as the key step in water oxidation catalysis. As a result, the two main pathways to accomplish this step, single-site water nucleophilic attack and O–O radical coupling, are discussed in detail and compared in terms of their potential use in photoelectrochemical cells for solar fuels generation.« less

  6. O–O bond formation in ruthenium-catalyzed water oxidation: single-site nucleophilic attack vs. O–O radical coupling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shaffer, David W.; Xie, Yan; Concepcion, Javier J.

    In this review we discuss at the mechanistic level the different steps involved in water oxidation catalysis with ruthenium-based molecular catalysts. We have chosen to focus on ruthenium-based catalysts to provide a more coherent discussion and because of the availability of detailed mechanistic studies for these systems but many of the aspects presented in this review are applicable to other systems as well. The water oxidation cycle has been divided in four major steps: water oxidative activation, O–O bond formation, oxidative activation of peroxide intermediates, and O 2 evolution. A significant portion of the review is dedicated to the O–Omore » bond formation step as the key step in water oxidation catalysis. As a result, the two main pathways to accomplish this step, single-site water nucleophilic attack and O–O radical coupling, are discussed in detail and compared in terms of their potential use in photoelectrochemical cells for solar fuels generation.« less

  7. O-O bond formation in ruthenium-catalyzed water oxidation: single-site nucleophilic attack vs. O-O radical coupling.

    PubMed

    Shaffer, David W; Xie, Yan; Concepcion, Javier J

    2017-10-16

    In this review we discuss at the mechanistic level the different steps involved in water oxidation catalysis with ruthenium-based molecular catalysts. We have chosen to focus on ruthenium-based catalysts to provide a more coherent discussion and because of the availability of detailed mechanistic studies for these systems but many of the aspects presented in this review are applicable to other systems as well. The water oxidation cycle has been divided in four major steps: water oxidative activation, O-O bond formation, oxidative activation of peroxide intermediates, and O 2 evolution. A significant portion of the review is dedicated to the O-O bond formation step as the key step in water oxidation catalysis. The two main pathways to accomplish this step, single-site water nucleophilic attack and O-O radical coupling, are discussed in detail and compared in terms of their potential use in photoelectrochemical cells for solar fuels generation.

  8. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  9. Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

    PubMed Central

    2015-01-01

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

  10. Non-competitive inhibition by active site binders.

    PubMed

    Blat, Yuval

    2010-06-01

    Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.

  11. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive materialmore » contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.« less

  12. Ni-Nanocluster Modified Black TiO2 with Dual Active Sites for Selective Photocatalytic CO2 Reduction.

    PubMed

    Billo, Tadesse; Fu, Fang-Yu; Raghunath, Putikam; Shown, Indrajit; Chen, Wei-Fu; Lien, Hsiang-Ting; Shen, Tzu-Hsien; Lee, Jyh-Fu; Chan, Ting-Shan; Huang, Kuo-You; Wu, Chih-I; Lin, M C; Hwang, Jih-Shang; Lee, Chih-Hao; Chen, Li-Chyong; Chen, Kuei-Hsien

    2018-01-01

    One of the key challenges in artificial photosynthesis is to design a photocatalyst that can bind and activate the CO 2 molecule with the smallest possible activation energy and produce selective hydrocarbon products. In this contribution, a combined experimental and computational study on Ni-nanocluster loaded black TiO 2 (Ni/TiO 2[Vo] ) with built-in dual active sites for selective photocatalytic CO 2 conversion is reported. The findings reveal that the synergistic effects of deliberately induced Ni nanoclusters and oxygen vacancies provide (1) energetically stable CO 2 binding sites with the lowest activation energy (0.08 eV), (2) highly reactive sites, (3) a fast electron transfer pathway, and (4) enhanced light harvesting by lowering the bandgap. The Ni/TiO 2[Vo] photocatalyst has demonstrated highly selective and enhanced photocatalytic activity of more than 18 times higher solar fuel production than the commercial TiO 2 (P-25). An insight into the mechanisms of interfacial charge transfer and product formation is explored. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

    PubMed

    Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2017-11-01

    The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

    PubMed

    Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

    2016-03-01

    An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures.

  15. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  16. An Evaluation of Subsurface Microbial Activity Conditional to Subsurface Temperature, Porosity, and Permeability at North American Carbon Sequestration Sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilson, B.; Mordensky, S.; Verba, Circe

    Several nations, including the United States, recognize global climate change as a force transforming the global ecosphere. Carbon dioxide (CO 2) is a greenhouse gas that contributes to the evolving climate. Reduction of atmospheric CO 2 levels is a goal for many nations and carbon sequestration which traps CO 2 in the Earth’s subsurface is one method to reduce atmospheric CO 2 levels. Among the variables that must be considered in developing this technology to a national scale is microbial activity. Microbial activity or biomass can change rock permeability, alter artificial seals around boreholes, and play a key role inmore » biogeochemistry and accordingly may determine how CO 2 is sequestered underground. Certain physical parameters of a reservoir found in literature (e.g., temperature, porosity, and permeability) may indicate whether a reservoir can host microbial communities. In order to estimate which subsurface formations may host microbes, this report examines the subsurface temperature, porosity, and permeability of underground rock formations that have high potential to be targeted for CO 2 sequestration. Of the 268 North American wellbore locations from the National Carbon Sequestration Database (NATCARB; National Energy and Technology Laboratory, 2015) and 35 sites from Nelson and Kibler (2003), 96 sequestration sites contain temperature data. Of these 96 sites, 36 sites have temperatures that would be favorable for microbial survival, 48 sites have mixed conditions for supporting microbial populations, and 11 sites would appear to be unfavorable to support microbial populations. Future studies of microbe viability would benefit from a larger database with more formation parameters (e.g. mineralogy, structure, and groundwater chemistry), which would help to increase understanding of where CO 2 sequestration could be most efficiently implemented.« less

  17. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  18. Discriminative structural approaches for enzyme active-site prediction.

    PubMed

    Kato, Tsuyoshi; Nagano, Nozomi

    2011-02-15

    Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

  19. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  20. Photogeneration of active formate decomposition catalysts to produce hydrogen from formate and water

    DOEpatents

    King, Jr., Allen D.; King, Robert B.; Sailers, III, Earl L.

    1983-02-08

    A process for producing hydrogen from formate and water by photogenerating an active formate decomposition catalyst from transition metal carbonyl precursor catalysts at relatively low temperatures and otherwise mild conditions is disclosed. Additionally, this process may be expanded to include the generation of formate from carbon monoxide and hydroxide such that the result is the water gas shift reaction.

  1. Ionizable side chains at catalytic active sites of enzymes.

    PubMed

    Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

    2012-05-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

  2. Star formation around active galactic nuclei

    NASA Technical Reports Server (NTRS)

    Keel, William C.

    1987-01-01

    Active galactic nuclei (Seyfert nuclei and their relatives) and intense star formation can both deliver substantial amounts of energy to the vicinity of a galactic nucleus. Many luminous nuclei have energetics dominated by one of these mechanisms, but detailed observations show that some have a mixture. Seeing both phenomena at once raises several interesting questions: (1) Is this a general property of some kinds of nuclei? How many AGNs have surround starbursts, and vice versa? (2) As in 1, how many undiscovered AGNs or starbursts are hidden by a more luminous instance of the other? (3) Does one cause the other, and by what means, or do both reflect common influences such as potential well shape or level of gas flow? (4) Can surrounding star formation tell us anything about the central active nuclei, such as lifetimes, kinetic energy output, or mechanical disturbance of the ISM? These are important points in the understanding of activity and star formation in galactic nuclei. Unfortunately, the observational ways of addressing them are as yet not well formulated. Some preliminary studies are reported, aimed at clarifying the issues involved in study of the relationships between stellar and nonstellar excitement in galactic nuclei.

  3. Human Topoisomerase I C-Terminal Domain Fragment Containing the Active Site Tyrosine is a Molten Globule: Implication for the Formation of Competent Productive Complex

    PubMed Central

    Punchihewa, Chandanamali; Dai, Jixun; Carver, Megan; Yang, Danzhou

    2007-01-01

    Human topoisomerase I (topo I) is an essential cellular enzyme that relaxes DNA supercoiling. The 6.3 kDa C-terminal domain of topo I contains the active site tyrosine (Tyr723) but lacks enzymatic activity by itself. Activity can be fully reconstituted when the C-terminal is associated with the 56 kDa core domain. Even though several crystal structures of topo I/DNA complexes are available, crystal structures of the free topo I protein or its individual domain fragments have been difficult to obtain. In this report we analyze the human topo I C-terminal domain structure using a variety of biophysical methods. Our results indicate that this fragment protein (topo6.3) appears to be in a molten globule state. It appears to have a native-like tertiary fold that contains a large population of α-helix secondary structure and extensive surface hydrophobic regions. Topo6.3 is known to be readily activated with the association of the topo I core domain, and the molten globule state of topo6.3 is likely to be an energy-favorable conformation for the free topo I C-terminal domain protein. The structural fluctuation and plasticity may represent an efficient mechanism in the topo I functional pathway, where the flexibility aids in the complementary association with the core domain and in the formation of a fully productive topo I complex. PMID:17434318

  4. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry uponmore » binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.« less

  5. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  6. Quantum mechanical design of enzyme active sites.

    PubMed

    Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

    2008-02-01

    The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

  7. Criteria for Formation of Active Personal Position of Schoolchildren

    ERIC Educational Resources Information Center

    Kunanbayeva, Magziya Sh.

    2016-01-01

    The article considers the problem and the importance of formation of the active personal position of schoolchildren. Active personal position is a complex concept, which includes the ability to a problem solution, the ability to work in a team, the ability to express his or her views. The formation of an active personal position at school is…

  8. Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

    PubMed Central

    Elbediwy, Ahmed; Zihni, Ceniz; Terry, Stephen J.; Clark, Peter

    2012-01-01

    Epithelial cell–cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell–cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin–capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics. PMID:22891260

  9. Thermal regime of active layer at two lithologically contrasting sites on James Ross Island, Antarctic Peninsula.

    NASA Astrophysics Data System (ADS)

    Hrbáček, Filip; Nývlt, Daniel; Láska, Kamil

    2016-04-01

    Antarctic Peninsula region (AP) represents one of the most rapidly warming parts of our planet in the last 50 years. Despite increasing research activities along both western and eastern sides of AP in last decades, there is still a lot of gaps in our knowledge relating to permafrost, active layer and its thermal and physical properties. This study brings new results of active layer monitoring on James Ross Island, which is the largest island in northern AP. Its northern part, Ulu Peninsula, is the largest ice-free area (more than 200 km2) in the region. Due its large area, we focused this study on sites located in different lithologies, which would affect local thermal regime of active layer. Study site (1) at Abernethy Flats area (41 m a.s.l.) lies ~7 km from northern coast. Lithologically is formed by disintegrated Cretaceous calcareous sandstones and siltstones of the Santa Marta Formation. Study site (2) is located at the northern slopes of Berry Hill (56 m a.s.l.), about 0.4 km from northern coastline. Lithology is composed of muddy to intermediate diamictites, tuffaceous siltstones to fine grained sandstones of the Mendel Formation. Data of air temperature at 2 meters above ground and the active layer temperatures at 75 cm deep profiles were obtained from both sites in period 1 January 2012 to 31 December 2014. Small differences were found when comparing mean air temperatures and active temperatures at 5 and 75 cm depth in the period 2012-2014. While the mean air temperatures varied between -7.7 °C and -7.0 °C, the mean ground temperatures fluctuated between -6.6 °C and -6.1 °C at 5 cm and -6.9 °C and -6.0 °C at 75 cm at Abernethy Flats and Berry Hill slopes respectively. Even though ground temperature differences along the profiles weren't pronounced during thawing seasons, the maximum active layer thickness was significantly larger at Berry Hill slopes (80 to 82 cm) than at Abernethy Flats (52 to 64 cm). We assume this differences are affected by

  10. Theoretical investigation of the role of clay edges in prebiotic peptide bond formation. II - Structures and thermodynamics of the activated complex species

    NASA Technical Reports Server (NTRS)

    Collins, Jack R.; Loew, Gilda H.; Luke, Brian T.; White, David H.

    1988-01-01

    Molecular orbital calculations are used to study amino acid activation by anhydride formation in neutral phosphates and in tetrahedral silicate and aluminate sites on clay edges. The results agree with previous ab initio studies of Luke et al. (1984) on the reactant species. Relative heats of formation of the anhydrides indicate the extent of anhydride formation to be the greatest for Al and the least for phosphate, which is the same order as the stability of hydrolysis.

  11. Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

    PubMed Central

    Shpakovski, G V; Berlin, Y A

    1984-01-01

    Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed. Images PMID:6091038

  12. Active sites of ligand-protected Au{sub 25} nanoparticle catalysts for CO{sub 2} electroreduction to CO

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alfonso, Dominic R., E-mail: alfonso@netl.doe.gov; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO{sub 2}) into CO at atomically precise negatively charged Au{sub 25}{sup −} nanoclusters. The studies showed CO{sub 2} conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO{sub 2} reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO{sub 2} reduction catalyst because formationmore » of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO{sub 2} reduction at gold nanocluster catalysts.« less

  13. Penetration Peg Formation and Invasive Hyphae Development Require Stage-Specific Activation of MoGTI1 in Magnaporthe oryzae.

    PubMed

    Li, Yang; Wang, Guanghui; Xu, Jin-Rong; Jiang, Cong

    2016-01-01

    The hemibiotrophic pathogen Magnaporthe oryzae causes one of the most destructive diseases in cultivated rice. Complex infection-related morphogenesis and production of various effectors are known to be important for successful colonization and disease development. In this study, we characterized the activation of the MoGTI1 transcription factor and its role in infection-related morphogenesis and effector gene expression. The Mogti1 mutant was nonpathogenic, although it was normal in appressorium formation and turgor generation. Close examination showed that Mogti1 was defective in penetration and growth of normal invasive hyphae. Deletion of MoGTI1 affected the expression of the majority of effector genes. The expression of MoGti1 appeared to be controlled by the Mps1 but not Pmk1 mitogen-activated protein kinase (MAPK), and the mps1 and Mogti1 mutants had similar phenotypes in plant infection and cell wall integrity defects. However, lack of MAPK phosphorylation sites and dispensability of the putative MAPK docking site suggested that MoGti1 is not a direct target of Mps1. Site-specific mutagenesis analyses showed that the putative protein kinase A phosphorylation site was not essential for localization of MoGti1 to the nucleus but important for its normal function. Although the cyclin-dependent kinase (CDK) phosphorylation site of MoGti1 is dispensable during vegetative growth and appressorium formation, the S77A mutation affected penetration and invasive growth. Localization of MoGti1(S77A)-green fluorescent protein to the nucleus in late stages of appressorium formation and during invasive growth was not observed, suggesting a stage-specific CDK phosphorylation of MoGti1. Overall, our data indicate that Mps1 may indirectly regulate the expression of MoGti1 in maintaining cell wall integrity, conidiation, and plant infection. MoGti1 is likely a stage-specific target of CDK and plays a crucial role in effector gene expression and morphogenesis related to the

  14. Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

    PubMed Central

    Fuentes, Eduardo; Caballero, Julio; Alarcón, Marcelo; Rojas, Armando; Palomo, Iván

    2014-01-01

    Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway. PMID:24598787

  15. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  16. Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

    PubMed

    Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

    1999-03-01

    The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

  17. KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation

    PubMed Central

    Sharma, Nishi R.; Majerciak, Vladimir; Kruhlak, Michael J.

    2017-01-01

    TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi’s sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection. PMID:29084250

  18. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific.

    PubMed

    Thune, I; Furberg, A S

    2001-06-01

    The association between physical activity and overall and site-specific cancer risk is elaborated in relation to whether any observed dose-response association between physical activity and cancer can be interpreted in terms of how much physical activity (type, intensity, duration, frequency) is needed to influence site- and gender-specific cancer risk. Observational studies were reviewed that have examined the independent effect of the volume of occupational physical activity (OPA) and/or leisure time physical activity (LPA) on overall and site-specific cancer risk. The evidence of cohort and case-control studies suggests that both leisure time and occupational physical activity protect against overall cancer risk, with a graded dose-response association suggested in both sexes. Confounding effects such as diet, body weight, and parity are often included as a covariate in the analyses, with little influence on the observed associations. A crude graded inverse dose-response association was observed between physical activity and colon cancer in 48 studies including 40,674 colon/colorectal cancer cases for both sexes. A dose-response effect of physical activity on colon cancer risk was especially observed, when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed as MET-hours per week. An observed inverse association with a dose-response relationship between physical activity and breast cancer was also identified in the majority of the 41 studies including 108,031 breast cancer cases. The dose-response relationship was in particular observed in case-control studies and supported by observations in cohort studies when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed by MET-hours per week. This association between physical activity and breast cancer risk is possibly dependent on age at exposure, age at diagnosis, menopausal status and other effect

  19. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-07

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

  20. NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region.

    PubMed Central

    Stamatoyannopoulos, J A; Goodwin, A; Joyce, T; Lowrey, C H

    1995-01-01

    The beta-like globin genes require the upstream locus control region (LCR) for proper expression. The active elements of the LCR coincide with strong erythroid-specific DNase I-hypersensitive sites (HSs). We have used 5' HS4 as a model to study the formation of these HSs. Previously, we identified a 101 bp element that is required for the formation of this HS. This element binds six proteins in vitro. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure. Images PMID:7828582

  1. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  2. Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

    PubMed

    Lui, Jennifer; Castelli, Lydia M; Pizzinga, Mariavittoria; Simpson, Clare E; Hoyle, Nathaniel P; Bailey, Kathryn L; Campbell, Susan G; Ashe, Mark P

    2014-11-06

    The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Vertical stratification of subsurface microbial community composition across geological formations at the Hanford Site.

    PubMed

    Lin, Xueju; Kennedy, David; Fredrickson, Jim; Bjornstad, Bruce; Konopka, Allan

    2012-02-01

    Microbial diversity in subsurface sediments at the Hanford Site 300 Area near Richland, Washington state (USA) was investigated by analysing 21 samples recovered from depths of 9-52 m. Approximately 8000 near full-length 16S rRNA gene sequences were analysed across geological strata that include a natural redox transition zone. These strata included the oxic coarse-grained Hanford formation, fine-grained oxic and anoxic Ringold Formation sediments, and the weathered basalt group. We detected 1233 and 120 unique bacterial and archaeal OTUs (operational taxonomic units at the 97% identity level) respectively. Microbial community structure and richness varied substantially across the different geological strata. Bacterial OTU richness (Chao1 estimator) was highest (> 700) in the upper Hanford formation, and declined to about 120 at the bottom of the Hanford formation. Just above the Ringold oxic-anoxic interface, richness was about 325 and declined to less than 50 in the deeper reduced zones. The deeper Ringold strata were characterized by a preponderance (c. 90%) of Proteobacteria. The bacterial community in the oxic sediments contained not only members of nine well-recognized phyla but also an unusually high proportion of three candidate divisions (GAL15, NC10 and SPAM). Additionally, 13 novel phylogenetic orders were identified within the Deltaproteobacteria, a clade rich in microbes that carry out redox transformations of metals that are important contaminants on the Hanford Site. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  4. Surface structural-chemical characterization of a single-site d0 heterogeneous arene hydrogenation catalyst having 100% active sites

    PubMed Central

    Williams, Linda A.; Guo, Neng; Motta, Alessandro; Delferro, Massimiliano; Fragalà, Ignazio L.; Miller, Jeffrey T.; Marks, Tobin J.

    2013-01-01

    Structural characterization of the catalytically significant sites on solid catalyst surfaces is frequently tenuous because their fraction, among all sites, typically is quite low. Here we report the combined application of solid-state 13C-cross-polarization magic angle spinning nuclear magnetic resonance (13C-CPMAS-NMR) spectroscopy, density functional theory (DFT), and Zr X-ray absorption spectroscopy (XAS) to characterize the adsorption products and surface chemistry of the precatalysts (η5-C5H5)2ZrR2 (R = H, CH3) and [η5-C5(CH3)5]Zr(CH3)3 adsorbed on Brønsted superacidic sulfated alumina (AlS). The latter complex is exceptionally active for benzene hydrogenation, with ∼100% of the Zr sites catalytically significant as determined by kinetic poisoning experiments. The 13C-CPMAS-NMR, DFT, and XAS data indicate formation of organozirconium cations having a largely electrostatic [η5-C5(CH3)5]Zr(CH3)2+···AlS− interaction with greatly elongated Zr···OAlS distances of ∼2.35(2) Å. The catalytic benzene hydrogenation cycle is stepwise understandable by DFT, and proceeds via turnover-limiting H2 delivery to surface [η5-C5(CH3)5]ZrH2(benzene)+···AlS− species, observable by solid-state NMR and XAS. PMID:23269836

  5. Direct instrumental identification of catalytically active surface sites

    NASA Astrophysics Data System (ADS)

    Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

    2017-09-01

    The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

  6. Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

    PubMed

    Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

    2012-01-01

    Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

  7. Atomically dispersed Ni(i) as the active site for electrochemical CO2 reduction

    NASA Astrophysics Data System (ADS)

    Yang, Hong Bin; Hung, Sung-Fu; Liu, Song; Yuan, Kaidi; Miao, Shu; Zhang, Liping; Huang, Xiang; Wang, Hsin-Yi; Cai, Weizheng; Chen, Rong; Gao, Jiajian; Yang, Xiaofeng; Chen, Wei; Huang, Yanqiang; Chen, Hao Ming; Li, Chang Ming; Zhang, Tao; Liu, Bin

    2018-02-01

    Electrochemical reduction of CO2 to chemical fuel offers a promising strategy for managing the global carbon balance, but presents challenges for chemistry due to the lack of effective electrocatalyst. Here we report atomically dispersed nickel on nitrogenated graphene as an efficient and durable electrocatalyst for CO2 reduction. Based on operando X-ray absorption and photoelectron spectroscopy measurements, the monovalent Ni(i) atomic center with a d9 electronic configuration was identified as the catalytically active site. The single-Ni-atom catalyst exhibits high intrinsic CO2 reduction activity, reaching a specific current of 350 A gcatalyst-1 and turnover frequency of 14,800 h-1 at a mild overpotential of 0.61 V for CO conversion with 97% Faradaic efficiency. The catalyst maintained 98% of its initial activity after 100 h of continuous reaction at CO formation current densities as high as 22 mA cm-2.

  8. Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site.

    PubMed

    Fetherolf, Morgan M; Boyd, Stefanie D; Taylor, Alexander B; Kim, Hee Jong; Wohlschlegel, James A; Blackburn, Ninian J; Hart, P John; Winge, Dennis R; Winkler, Duane D

    2017-07-21

    Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  10. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  11. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  12. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  13. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  14. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

  15. Formation of reactive oxygen by N2O decomposition over binuclear cationic sites of Fe-ferrierite zeolite: Periodic DFT + U study

    NASA Astrophysics Data System (ADS)

    Avdeev, Vasilii I.; Bedilo, Alexander F.

    2018-03-01

    The electronic nature of sites over Fe-ferrierite zeolite stabilizing active α-oxygen is analyzed by the periodic DFT + U approach. It is shown that two antiferromagnetically coupled Fe2+ cations with bridging OH-bonds form a stable bi-nuclear site of the [Fe2+<2OH>Fe2+] doped FER complex. Frontier orbitals of this complex populated by two electrons with minority spins are localized in the bandgap. As a result, [Fe2+<2OH>Fe2+] unit acquires the properties of a binuclear Lewis acid dipolarophile for 1,3-dipole N2O. First reaction step of N2O decomposition follows the Huisgen‧s concept of the 1,3-dipolar cycloaddition concept followed by the formation of reactive oxygen species Fesbnd O.

  16. Microwave-induced activation of additional active edge sites on the MoS2 surface for enhanced Hg0 capture

    NASA Astrophysics Data System (ADS)

    Zhao, Haitao; Mu, Xueliang; Yang, Gang; Zheng, Chengheng; Sun, Chenggong; Gao, Xiang; Wu, Tao

    2017-10-01

    In recent years, significant effort has been made in the development of novel materials for the removal of mercury from coal-derived flue gas. In this research, microwave irradiation was adopted to induce the creation of additional active sites on the MoS2 surface. The results showed that Hg0 capture efficiency of the adsorbent containing MoS2 nanosheets being microwave treated was as high as 97%, while the sample prepared via conventional method only showed an efficiency of 94% in its first 180 min testing. After the adsorbent was treated by microwave irradiation for 3 more times, its mercury removal efficiency was still noticeably higher than that of the sample prepared via conventional method. Characterization of surface structure of the MoS2 containing material together with DFT study further revealed that the (001) basal planes of MoS2 crystal structure were cracked into (100) edge planes (with an angle of approximately 75°) under microwave treatment, which subsequently resulted in the formation of additional active edge sites on the MoS2 surface and led to the improved performance on Hg0 capture.

  17. Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis.

    PubMed

    Bolten, Marcel; Vahlensieck, Christian; Lipp, Colette; Leibundgut, Marc; Ban, Nenad; Weber-Ban, Eilika

    2017-03-10

    Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Comment on "Active sites for CO2 hydrogenation to methanol on Cu/ZnO catalysts".

    PubMed

    Nakamura, Junji; Fujitani, Tadahiro; Kuld, Sebastian; Helveg, Stig; Chorkendorff, Ib; Sehested, Jens

    2017-09-01

    Kattel et al (Reports, 24 March 2017, p. 1296) report that a zinc on copper (Zn/Cu) surface undergoes oxidation to zinc oxide/copper (ZnO/Cu) during carbon dioxide (CO 2 ) hydrogenation to methanol and conclude that the Cu-ZnO interface is the active site for methanol synthesis. Similar experiments conducted two decades ago by Fujitani and Nakamura et al demonstrated that Zn is attached to formate rather than being fully oxidized. Copyright © 2017, American Association for the Advancement of Science.

  19. Greater Bone Formation Induction Occurred in Aged than Young Cancellous Bone Sites

    NASA Technical Reports Server (NTRS)

    Ke, H. Z.; Jee, W. S. S.; Ito, H.; Setterberg, R. B.; Li, M.; Lin, B. Y.; Liang, X. G.; Ma, Y. F.

    1993-01-01

    We have determined the differences in the effects of continual prostaglandin E(sub 2) (PGE(sub 2) treatment in aged (non-growing) and young (growing) cancellous bone sites in 7-month-old Sprague-Dawley rats. The sites involved are the aged distal tibial metaphysis (DTM) with a closed epiphysis and the young proximal tibial metaphysis (PTM) with a slow growing, open epiphysis. The study involved rats treated with 0, 1, 3 or 6 mg PGE(sub 2)/kg/d for 60, 120 and 180 days. Static and dynamic histomorphometry of percent trabecular area, and tissue-referent bone formation rate (BFR/TV) were determined in both DTM and PTM. In pretreatment controls, the secondary spongiosa of the two metaphyses contain the same amount of cancellous bone (11% in DTM vs. 13% in PTM), but markedly less bone formation in DTM (0.6%/y in DTM vs. 41.5%/y in PTM). After 60 days of 6 mg PGE(sub 2)/kg/d treatment, %Tb.Ar was increased 607% in DTM and 199% in PTM, BFR/TV was increased to nearly 14 fold in DTM and only 5 fold in PTM. These results indicated the aged metaphysis of the DTM was much more responsive to PGE(sub 2) treatment than young, growing metaphysis of the PTM. The results of 120 and 180 days treatment did not significantly differ from 60 days treatment in both sites, indicating that the effect of continuous daily PGE2 treatment were in equilibrium after 60 days. We concluded that aged metaphysis was much more responsive to PGE(sub 2) treatment than young growing metaphysis.

  20. Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

    PubMed

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

  1. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  2. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  3. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  4. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  5. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

  6. Antibody-catalyzed benzoin oxidation as a mechanistic probe for nucleophilic catalysis by an active site lysine.

    PubMed

    Sklute, Genia; Oizerowich, Rachel; Shulman, Hagit; Keinan, Ehud

    2004-05-03

    Aldolase antibody 24H6, which was obtained by reactive immunization against a 1,3-diketone hapten, is shown to catalyze additional reactions, including H/D exchange and oxidation reactions. Comparison of the H/D exchange reaction at the alpha-position of a wide range of aldehydes and ketones by 24H6 and by other aldolase antibodies, such as 38C2, pointed at the significantly larger size of the 24H6 active site. This property allowed for the catalysis of the oxidation of substituted benzoins to benzils by potassium ferricyanide. This reaction was used as a mechanistic probe to learn about the initial steps of the 24H6-catalyzed aldol condensation reaction. The Hammett correlation (rho=4.7) of log(k(cat)) versus the substituent constant, sigma, revealed that the reaction involves rapid formation of a Schiff base intermediate from the ketone and an active site lysine residue. The rate-limiting step in this oxidation reaction is the conversion of the Schiff base to an enamine intermediate. In addition, linear correlation (rho=3.13) was found between log(K(M)) and sigma, indicating that electronic rather than steric factors are dominant in the antibody-substrate binding phenomenon and confirming that the reversible formation of a Schiff base intermediate comprises part of the substrate-binding mechanism.

  7. Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation

    PubMed Central

    Whalley, Helen J.; Porter, Andrew P.; Diamantopoulou, Zoi; White, Gavin R. M.; Castañeda-Saucedo, Eduardo; Malliri, Angeliki

    2015-01-01

    Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the development of Eg5 inhibitors as cancer therapeutics. PMID:26078008

  8. PPAR-γ activation by Tityus serrulatus venom regulates lipid body formation and lipid mediator production.

    PubMed

    Zoccal, Karina Furlani; Paula-Silva, Francisco Wanderley Garcia; Bitencourt, Claudia da Silva; Sorgi, Carlos Artério; Bordon, Karla de Castro Figueiredo; Arantes, Eliane Candiani; Faccioli, Lúcia Helena

    2015-01-01

    Tityus serrulatus venom (TsV) consists of numerous peptides with different physiological and pharmacological activities. Studies have shown that scorpion venom increases pro-inflammatory cytokine production, contributing to immunological imbalance, multiple organ dysfunction, and patient death. We have previously demonstrated that TsV is a venom-associated molecular pattern (VAMP) recognized by TLRs inducing intense inflammatory reaction through the production of pro-inflammatory cytokines and arachidonic acid-derived lipid mediators prostaglandin (PG)E2 and leukotriene (LT)B4. Lipid bodies (LBs) are potential sites for eicosanoid production by inflammatory cells. Moreover, recent studies have shown that the peroxisome proliferator-activated receptor gamma (PPAR-γ) is implicated in LB formation and acts as an important modulator of lipid metabolism during inflammation. In this study, we used murine macrophages to evaluate whether the LB formation induced by TsV after TLR recognition correlates with lipid mediator generation by macrophages and if it occurs through PPAR-γ activation. We demonstrate that TsV acts through TLR2 and TLR4 stimulation and PPAR-γ activation to induce LB formation and generation of PGE2 and LTB4. Our data also show that PPAR-γ negatively regulates the pro-inflammatory NF-κB transcription factor. Based on these results, we suggest that during envenomation, LBs constitute functional organelles for lipid mediator production through signaling pathways that depend on cell surface and nuclear receptors. These findings point to the inflammatory mechanisms that might also be triggered during human envenomation by TsV. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

    PubMed Central

    Dreveny, Ingrid; Kratky, Christoph; Gruber, Karl

    2002-01-01

    The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction—the enantiospecific formation of α-hydroxynitriles—is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C–C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident. PMID:11790839

  10. The role of beaded activated carbon's pore size distribution on heel formation during cyclic adsorption/desorption of organic vapors.

    PubMed

    Jahandar Lashaki, Masoud; Atkinson, John D; Hashisho, Zaher; Phillips, John H; Anderson, James E; Nichols, Mark

    2016-09-05

    The effect of activated carbon's pore size distribution (PSD) on heel formation during adsorption of organic vapors was investigated. Five commercially available beaded activated carbons (BAC) with varying PSDs (30-88% microporous) were investigated. Virgin samples had similar elemental compositions but different PSDs, which allowed for isolating the contribution of carbon's microporosity to heel formation. Heel formation was linearly correlated (R(2)=0.91) with BAC micropore volume; heel for the BAC with the lowest micropore volume was 20% lower than the BAC with the highest micropore volume. Meanwhile, first cycle adsorption capacities and breakthrough times correlated linearly (R(2)=0.87 and 0.93, respectively) with BAC total pore volume. Micropore volume reduction for all BACs confirmed that heel accumulation takes place in the highest energy pores. Overall, these results show that a greater portion of adsorbed species are converted into heel on highly microporous adsorbents due to higher share of high energy adsorption sites in their structure. This differs from mesoporous adsorbents (low microporosity) in which large pores contribute to adsorption but not to heel formation, resulting in longer adsorbent lifetime. Thus, activated carbon with high adsorption capacity and high mesopore fraction is particularly desirable for organic vapor application involving extended adsorption/regeneration cycling. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Computer simulation of the active site of human serum cholinesterase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positivemore » electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.« less

  12. Quenching of the star formation activity in cluster galaxies

    NASA Astrophysics Data System (ADS)

    Boselli, A.; Roehlly, Y.; Fossati, M.; Buat, V.; Boissier, S.; Boquien, M.; Burgarella, D.; Ciesla, L.; Gavazzi, G.; Serra, P.

    2016-11-01

    We study the star formation quenching mechanism in cluster galaxies by fitting the spectral energy distribution (SED) of the Herschel Reference Survey, a complete volume-limited K-band-selected sample of nearby galaxies including objects in different density regions, from the core of the Virgo cluster to the general field. The SEDs of the target galaxies were fitted using the CIGALE SED modelling code. The truncated activity of cluster galaxies was parametrised using a specific star formation history with two free parameters, the quenching age QA and the quenching factor QF. These two parameters are crucial for the identification of the quenching mechanism, which acts on long timescales when starvation processes are at work, but is rapid and efficient when ram pressure occurs. To be sensitive to an abrupt and recent variation of the star formation activity, we combined twenty photometric bands in the UV to far-infrared in a new way with three age-sensitive Balmer line absorption indices extracted from available medium-resolution (R 1000) integrated spectroscopy and with Hα narrow-band imaging data. The use of a truncated star formation history significantly increases the quality of the fit in HI-deficient galaxies of the sample, that is to say, in those objects whose atomic gas content has been removed during the interaction with the hostile cluster environment. The typical quenching age of the perturbed late-type galaxies is QA ≲ 300 Myr whenever the activity of star formation is reduced by 50% < QF ≤ 80% and QA ≲ 500 Myr for QF > 80%, while that of the quiescent early-type objects is QA ≃ 1-3 Gyr. The fraction of late-type galaxies with a star formation activity reduced by QF > 80% and with an HI-deficiency parameter HI-def > 0.4 drops by a factor of 5 from the inner half virial radius of the Virgo cluster (R/Rvir < 0.5), where the hot diffuse X-ray emitting gas of the cluster is located, to the outer regions (R/Rvir > 4). The efficient quenching of the

  13. Telomere formation on macronuclear chromosomes of Oxytricha trifallax and O. fallax: alternatively processed regions have multiple telomere addition sites

    PubMed Central

    Williams, Kevin R; Doak, Thomas G; Herrick, Glenn

    2002-01-01

    Background Ciliates employ massive chromatid breakage and de novo telomere formation during generation of the somatic macronucleus. Positions flanking the 81-MAC locus are reproducibly cut. But those flanking the Common Region are proposed to often escape cutting, generating three nested macronuclear chromosomes, two retaining "arms" still appended to the Common Region. Arm-distal positions must differ (in cis) from the Common Region flanks. Results The Common-Region-flanking positions also differ from the arm-distal positions in that they are "multi-TAS" regions: anchored PCR shows heterogeneous patterns of telomere addition sites, but arm-distal sites do not. The multi-TAS patterns are reproducible, but are sensitive to the sequence of the allele being processed. Thus, random degradation following chromatid cutting does not create this heterogeneity; these telomere addition sites also must be dictated by cis-acting sequences. Conclusions Most ciliates show such micro-heterogeneity in the precise positions of telomere addition sites. Telomerase is believed to be tightly associated with, and act in concert with, the chromatid-cutting nuclease: heterogeneity must be the result of intervening erosion activity. Our "weak-sites" hypothesis explains the correlation between alternative chromatid cutting at the Common Region boundaries and their multi-TAS character: when the chromatid-breakage machine encounters either a weak binding site or a weak cut site at these regions, then telomerase dissociates prematurely, leaving the new end subject to erosion by an exonuclease, which pauses at cis-acting sequences; telomerase eventually heals these resected termini. Finally, we observe TAS positioning influenced by trans-allelic interactions, reminiscent of transvection. PMID:12199911

  14. Surface formation, preservation, and history of low-porosity crusts at the WAIS Divide site, West Antarctica

    NASA Astrophysics Data System (ADS)

    Fegyveresi, John M.; Alley, Richard B.; Muto, Atsuhiro; Orsi, Anaïs J.; Spencer, Matthew K.

    2018-01-01

    Observations at the West Antarctic Ice Sheet (WAIS) Divide site show that near-surface snow is strongly altered by weather-related processes such as strong winds and temperature fluctuations, producing features that are recognizable in the deep ice core. Prominent glazed surface crusts develop frequently at the site during summer seasons. Surface, snow pit, and ice core observations made in this study during summer field seasons from 2008-2009 to 2012-2013, supplemented by automated weather station (AWS) data with short- and longwave radiation sensors, revealed that such crusts formed during relatively low-wind, low-humidity, clear-sky periods with intense daytime sunshine. After formation, such glazed surfaces typically developed cracks in a polygonal pattern likely from thermal contraction at night. Cracking was commonest when several clear days occurred in succession and was generally followed by surface hoar growth; vapor escaping through the cracks during sunny days may have contributed to the high humidity that favored nighttime formation of surface hoar. Temperature and radiation observations show that daytime solar heating often warmed the near-surface snow above the air temperature, contributing to upward mass transfer, favoring crust formation from below, and then surface hoar formation. A simple surface energy calculation supports this observation. Subsequent examination of the WDC06A deep ice core revealed that crusts are preserved through the bubbly ice, and some occur in snow accumulated during winters, although not as commonly as in summertime deposits. Although no one has been on site to observe crust formation during winter, it may be favored by greater wintertime wind packing from stronger peak winds, high temperatures and steep temperature gradients from rapid midwinter warmings reaching as high as -15 °C, and perhaps longer intervals of surface stability. Time variations in crust occurrence in the core may provide paleoclimatic

  15. De novo active sites for resurrected Precambrian enzymes

    NASA Astrophysics Data System (ADS)

    Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

    2017-07-01

    Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

  16. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    PubMed Central

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

  17. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    NASA Astrophysics Data System (ADS)

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

  18. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  19. Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates.

    PubMed

    Devine, Erin L; Theobald, Douglas L; Oprian, Daniel D

    2016-08-30

    The visual pigment rhodopsin is a G protein-coupled receptor that covalently binds its retinal chromophore via a Schiff base linkage to an active-site Lys residue in the seventh transmembrane helix. Although this residue is strictly conserved among all type II retinylidene proteins, we found previously that the active-site Lys in bovine rhodopsin (Lys296) can be moved to three other locations (G90K, T94K, S186K) while retaining the ability to form a pigment with retinal and to activate transducin in a light-dependent manner [ Devine et al. ( 2013 ) Proc. Natl. Acad. Sci. USA 110 , 13351 - 13355 ]. Because the active-site Lys is not functionally constrained to be in helix seven, it is possible that it could relocate within the protein, most likely via an evolutionary intermediate with two active-site Lys. Therefore, in this study we characterized potential evolutionary intermediates with two Lys in the active site. Four mutant rhodopsins were prepared in which the original Lys296 was left untouched and a second Lys residue was substituted for G90K, T94K, S186K, or F293K. All four constructs covalently bind 11-cis-retinal, form a pigment, and activate transducin in a light-dependent manner. These results demonstrate that rhodopsin can tolerate a second Lys in the retinal binding pocket and suggest that an evolutionary intermediate with two Lys could allow migration of the Schiff base Lys to a position other than the observed, highly conserved location in the seventh TM helix. From sequence-based searches, we identified two groups of natural opsins, insect UV cones and neuropsins, that contain Lys residues at two positions in their active sites and also have intriguing spectral similarities to the mutant rhodopsins studied here.

  20. Synthesis of Zn-MOF incorporating titanium-hydrides as active sites binding H{sub 2} molecules

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Jongsik, E-mail: jkim40@nd.edu; Ok Kim, Dong; Wook Kim, Dong

    2015-10-15

    This paper describes the synthetic effort for a Zn-MOF imparting Ti-H as a preferential binding site potentially capturing H{sub 2} molecules via Kubas-type interaction. The formation mechanism of Ti-H innate to the final material was potentially demonstrated to follow a radical dissociation rather than a β-hydrogen elimination and a C-H reductive elimination. - Graphical abstract: This study details the synthesis and the formation mechanism of Zn-MOF adsorbent site-isolating TiH{sub 3} that can potentially capture H{sub 2} molecules via Kubas-binding mechanism. - Highlights: • OH-functionalized Zn-MOF was employed as a reactive template to site-isolate TiH{sub 3}. • This MOF was post-syntheticallymore » modified using a tetracyclohexyl titanium (IV). • This intermediate was hydrogenolyzed to change ligand from cyclohexyl to hydride. • Formation mechanism of TiH{sub 3} was investigated via two control GC–MS experiments. • Final Zn-MOF potentially site-isolating TiH{sub 3} species was used as a H{sub 2} adsorbent.« less

  1. Influence of the Heterogeneous Nucleation Sites on the Kinetics of Intermetallic Phase Formation in Aged Duplex Stainless Steel

    NASA Astrophysics Data System (ADS)

    Melo, Elis Almeida; Magnabosco, Rodrigo

    2017-11-01

    The aim of this work is to study the influence of the heterogeneous nucleation site quantity, observed in different ferrite and austenite grain size samples, on the phase transformations that result in intermetallic phases in a UNS S31803 duplex stainless steel (DSS). Solution treatment was conducted for 1, 24, 96, or 192 hours at 1373 K (1100 °C) to obtain different ferrite and austenite grain sizes. After solution treatment, isothermal aging treatments for 5, 8, 10, 20, 30, or 60 minutes at 1123 K (850 °C) were performed to verify the influence of different amounts of heterogeneous nucleation sites in the kinetics of intermetallic phase formation. The sample solution treated for 1 hour, with the highest surface area between matrix phases, was the one that presented, after 60 minutes at 1123 K (850 °C), the smaller volume fraction of ferrite (indicative of greater intermetallic phase formation), higher volume of sigma (that was present in coral-like and compact morphologies), and chi phase. It was not possible to identify which was the first nucleated phase, sigma or chi. It was also observed that the phase formation kinetics is higher for the sample solution treated for 1 hour. It was evidenced that, from a certain moment on, the chi phase begins to be consumed due to the sigma phase formation, and the austenite/ferrite interface presents higher S V for all solution treatment times. It was also observed that intermetallic phases form preferably in austenite-ferrite interfaces, although the higher occupation rate occurs at triple junction ferrite-ferrite-ferrite. It was verified that there was no saturation of nucleation sites in any interface type nor triple junction, and the equilibrium after 1 hour of aging at 1123 K (850 °C) was not achieved. It was then concluded that sigma phase formation is possibly controlled by diffusional processes, without saturation of nucleation sites.

  2. Insights into site formation at Rose Cottage Cave, South Africa, based on the analysis of sediment peels

    NASA Astrophysics Data System (ADS)

    Kloos, Peter; Miller, Christopher E.; Kritikakis, Panagiotis; Wadley, Lyn

    2016-04-01

    Rose Cottage Cave (RCC), in South Africa, has been a key site for explaining the origins of modern human behaviour and movement of early modern humans out of Africa. Nine sediment peels were made previously from the profile sections, preserving original materials that provide a record of cultural and environmental change during the late Pleistocene and Holocene. Here, we present the preliminary results of the study of the RCC sediment peels which aims to investigate site formation processes and the implications for site interpretation. Methods used include micromorphology and Fourier Transform Infrared spectroscopy coupled with detailed observations of the peels. The predominance of geogenic processes is demonstrated by the abundance of silt- and sand-sized quartz grains, which entered the site primarily through a crevice at the back of the cave. RCC lacks rich anthropogenic deposits as noted at other Middle Stone Age sites in southern Africa, but anthropogenic input to the sediment is indicated by the presence of charcoal, burnt bone, lithic fragments, fat-derived char and ashes. Clay coating fragments and chaotic microstructures demonstrate that bioturbation and colluvial reworking homogenised much of the deposit and may explain the absence of preserved bedding and rarity of combustion features. Downward movement of water through the sequence, indicated by clay coatings, is the likely cause for poor bone preservation and near lack of ashes at the site, as well as fluctuations in dose rate that have complicated luminescence dating studies. Evidence for diagenesis at the site is in the form of secondary apatite and gypsum. Sedimentary structures such as channel lag deposits and (silt and sand) laminae observed in peels dating between 60 and 35 ka BP suggest a high-energy sedimentary environment, which experienced flooding events that eroded underlying deposits and deposited large volumes of sediment. This explains why some of the post-Howiesons Poort layers contain

  3. Spectroscopic insights into the nature of active sites in iron–nitrogen–carbon electrocatalysts for oxygen reduction in acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jia, Qingying; Ramaswamy, Nagappan; Tylus, Urszula

    -blocking effect. Moreover, a highly active MOF-based catalyst without Fe–N moieties was developed, and the active sites were identified as nitrogen-doped carbon fibers with embedded iron particles that are not directly involved in the oxygen reduction pathway. The high ORR activity and durability of catalysts involving this second site, as demonstrated in fuel cell, are attributed to the high density of active sites and the elimination or reduction of Fenton-type processes. The latter are initiated by hydrogen peroxide but are known to be accelerated by iron ions exposed to the surface, resulting in the formation of damaging free-radicals.« less

  4. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

  5. NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

    PubMed Central

    Dunn, Michael F.

    2013-01-01

    crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227

  6. Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes.

    PubMed

    Zastrow, Melissa L; Pecoraro, Vincent L

    2013-04-17

    While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

  7. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading ofmore » vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.« less

  8. U.S. Department of Energy's site screening, site selection, and initial characterization for storage of CO2 in deep geological formations

    USGS Publications Warehouse

    Rodosta, T.D.; Litynski, J.T.; Plasynski, S.I.; Hickman, S.; Frailey, S.; Myer, L.

    2011-01-01

    The U.S. Department of Energy (DOE) is the lead Federal agency for the development and deployment of carbon sequestration technologies. As part of its mission to facilitate technology transfer and develop guidelines from lessons learned, DOE is developing a series of best practice manuals (BPMs) for carbon capture and storage (CCS). The "Site Screening, Site Selection, and Initial Characterization for Storage of CO2 in Deep Geological Formations" BPM is a compilation of best practices and includes flowchart diagrams illustrating the general decision making process for Site Screening, Site Selection, and Initial Characterization. The BPM integrates the knowledge gained from various programmatic efforts, with particular emphasis on the Characterization Phase through pilot-scale CO2 injection testing of the Validation Phase of the Regional Carbon Sequestration Partnership (RCSP) Initiative. Key geologic and surface elements that suitable candidate storage sites should possess are identified, along with example Site Screening, Site Selection, and Initial Characterization protocols for large-scale geologic storage projects located across diverse geologic and regional settings. This manual has been written as a working document, establishing a framework and methodology for proper site selection for CO2 geologic storage. This will be useful for future CO2 emitters, transporters, and storage providers. It will also be of use in informing local, regional, state, and national governmental agencies of best practices in proper sequestration site selection. Furthermore, it will educate the inquisitive general public on options and processes for geologic CO2 storage. In addition to providing best practices, the manual presents a geologic storage resource and capacity classification system. The system provides a "standard" to communicate storage and capacity estimates, uncertainty and project development risk, data guidelines and analyses for adequate site characterization, and

  9. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes

  10. Base Mechanism to the Hydrolysis of Phosphate Triester Promoted by the Cd2+/Cd2+ Active site of Phosphotriesterase: A Computational Study.

    PubMed

    Chagas, Marcelo A; Pereira, Eufrásia S; Godinho, Marina P B; Da Silva, Júlio Cosme S; Rocha, Willian R

    2018-05-21

    In the present work, density functional theory (DFT) calculations at the B3LYP/6-31+G(d) and including dispersion effects were used to investigate the hydrolysis of paraoxon, using a cluster model of the active site of Cd 2+ /Cd 2+ -phosphotriesterase (PTE) from Pseudomonas diminuta. The mechanism proposed here consist of (i) Exchange of the coordinated water molecule and coordination of the substrate to the more solvent exposed Cd β center in monodentate fashion, (ii) protonation of the μ-hydroxo bridge by the uncoordinated water molecule and in situ formation of the nucleophile, (iii) formation of a pentacoordinate intermediate with significant bond breaking to the leaving group and bond formation to the nucleophile, and (iv) protonation of the Asp301 residue and restoration of the active site through the coordination of another water molecule of the medium. The water molecules initially coordinated to the active site play a crucial role in stabilizing the transition states and the pentacoordinate intermediate. The reaction takes place in a two-step (A N + D N ) mechanism, with energy barriers of 12.9 and 1.9 kcal/mol for the first and second steps, respectively, computed at the B3LYP-D3/6-311++G(2d,2p) level of theory, in excellent agreement with the experimental findings. Dispersion effects alone contribute to diminish the energy barriers as much as 26%. The base mechanism for the Cd 2+ /Cd 2+ -PTE proposed here, in conjunction with the agreement found with the experimental energetic value for the energy barrier, makes it a consistent and kinetically viable mechanistic proposal for the hydrolysis of phosphate triesters promoted by the Cd 2+ substituted PTE enzyme.

  11. Chemical signal activation of an organocatalyst enables control over soft material formation.

    PubMed

    Trausel, Fanny; Maity, Chandan; Poolman, Jos M; Kouwenberg, D S J; Versluis, Frank; van Esch, Jan H; Eelkema, Rienk

    2017-10-12

    Cells can react to their environment by changing the activity of enzymes in response to specific chemical signals. Artificial catalysts capable of being activated by chemical signals are rare, but of interest for creating autonomously responsive materials. We present an organocatalyst that is activated by a chemical signal, enabling temporal control over reaction rates and the formation of materials. Using self-immolative chemistry, we design a deactivated aniline organocatalyst that is activated by the chemical signal hydrogen peroxide and catalyses hydrazone formation. Upon activation of the catalyst, the rate of hydrazone formation increases 10-fold almost instantly. The responsive organocatalyst enables temporal control over the formation of gels featuring hydrazone bonds. The generic design should enable the use of a large range of triggers and organocatalysts, and appears a promising method for the introduction of signal response in materials, constituting a first step towards achieving communication between artificial chemical systems.Enzymes regulated by chemical signals are common in biology, but few such artificial catalysts exist. Here, the authors design an aniline catalyst that, when activated by a chemical trigger, catalyses formation of hydrazone-based gels, demonstrating signal response in a soft material.

  12. Characteristics of formation and growth of atmospheric nanoparticles observed at four regional background sites in Korea

    NASA Astrophysics Data System (ADS)

    Kim, Yumi; Kim, Sang-Woo; Yoon, Soon-Chang; Park, Jin-Soo; Lim, Jae-Hyun; Hong, Jihyung; Lim, Han-Cheol; Ryu, Jegyu; Lee, Chul-Kyu; Heo, Bok-Haeng

    2016-02-01

    Measurements of the number concentration and size distribution of atmospheric nanoparticles were conducted at four sites on the west coast of the Korean Peninsula by using identical scanning mobility particle sizers (SMPSs) in October 2012. The new particle formation and subsequent growth (NPF) of atmospheric nanoparticles, which were identified by the cyclostationary empirical orthogonal function (CSEOF) analysis technique, was observed on 11 out of 21 days at the Baengnyeong-do Comprehensive Monitoring Observatory (BCMO); and on 10 out of 21 days at the Korea Global Atmosphere Watch Center (KGAWC) from October 9 to 29, 2012. We also observed NPF events for 9 out of 21 days at both the Gosan Climate Observatory (GCO) and the Jeju Comprehensive Monitoring Observatory (JCMO). During the study period, NPF was simultaneously observed for five days at all four sites, which indicates that the NPF event had a spatial extent of at least 540 km. A cold, dry and cloud-free continental air mass originated from northern China, formed favorable environmental conditions (e.g., increasing solar insolation at the surface) on simultaneous NPF at the four sites. These synoptic weather patterns were closely associated with an extraordinary typhoon passing over the south of Japan. The mean values of particle formation rates at BCMO (1.26 cm- 3 s- 1) and KGAWC (1.49 cm- 3 s- 1) were relatively higher than those at GCO (0.39 cm- 3 s- 1) and JCMO (0.74 cm- 3 s- 1), however, the growth rate showed a similar level among four sites. An increase in the spatial homogeneity and inter-site correlation of atmospheric particles among the four sites was apparent for small particles (diameter < 30 nm) on simultaneous NPF event days.

  13. Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation.

    PubMed Central

    Huber, M C; Bosch, F X; Sippel, A E; Bonifer, C

    1994-01-01

    The complete chicken lysozyme gene locus is expressed copy number dependently and at a high level in macrophages of transgenic mice. Gene expression independent of genomic position can only be achieved by the concerted action of all cis regulatory elements located on the lysozyme gene domain. Position independency of expression is lost if one essential cis regulatory region is deleted. Here we compared the DNase I hypersensitive site (DHS) pattern formed on the chromatin of position independently and position dependently expressed transgenes in order to assess the influence of deletions within the gene domain on active chromatin formation. We demonstrate, that in position independently expressed transgene all DHSs are formed with the authentic relative frequency on all genes. This is not the case for position dependently expressed transgenes. Our results show that the formation of a DHS during cellular differentiation does not occur autonomously. In case essential regulatory elements of the chicken lysozyme gene domain are lacking, the efficiency of DHS formation on remaining cis regulatory elements during myeloid differentiation is reduced and influenced by the chromosomal position. Hence, no individual regulatory element on the lysozyme domain is capable of organizing the chromatin structure of the whole locus in a dominant fashion. Images PMID:7937145

  14. All the catalytic active sites of MoS 2 for hydrogen evolution

    DOE PAGES

    Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

    2016-11-29

    MoS 2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS 2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated tomore » be 7.5 s –1 (65–75 mV/dec), 3.2 s –1 (65–85 mV/dec), and 0.1 s –1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS 2 is higher than that in low crystalline quality MoS 2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

  15. Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

    DOE PAGES

    Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping; ...

    2017-07-27

    Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

  16. Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping

    Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

  17. Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity.

    PubMed

    Chen, Zhi-Wei; Datta, Saumen; Dubois, Jennifer L; Klinman, Judith P; Mathews, F Scott

    2010-08-31

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  18. Bridge-bonded formate: active intermediate or spectator species in formic acid oxidation on a Pt film electrode?

    PubMed

    Chen, Y-X; Heinen, M; Jusys, Z; Behm, R J

    2006-12-05

    We present and discuss the results of an in situ IR study on the mechanism and kinetics of formic acid oxidation on a Pt film/Si electrode, performed in an attenuated total reflection (ATR) flow cell configuration under controlled mass transport conditions, which specifically aimed at elucidating the role of the adsorbed bridge-bonded formates in this reaction. Potentiodynamic measurements show a complex interplay between formation and desorption/oxidation of COad and formate species and the total Faradaic current. The notably faster increase of the Faradaic current compared to the coverage of bridge-bonded formate in transient measurements at constant potential, but with different formic acid concentrations, reveals that adsorbed formate decomposition is not rate-limiting in the dominant reaction pathway. If being reactive intermediate at all, the contribution of formate adsorption/decomposition to the reaction current decreases with increasing formic acid concentration, accounting for at most 15% for 0.2 M DCOOH at 0.7 VRHE. The rapid build-up/removal of the formate adlayer and its similarity with acetate or (bi-)sulfate adsorption/desorption indicate that the formate adlayer coverage is dominated by a fast dynamic adsorption-desorption equilibrium with the electrolyte, and that formate desorption is much faster than its decomposition. The results corroborate the proposal of a triple pathway reaction mechanism including an indirect pathway, a formate pathway, and a dominant direct pathway, as presented previously (Chen, Y. X.; et al. Angew. Chem. Int. Ed. 2006, 45, 981), in which adsorbed formates act as a site-blocking spectator in the dominant pathway rather than as an active intermediate.

  19. Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

    PubMed Central

    Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

    2014-01-01

    Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

  20. Using an ACTIVE teaching format versus a standard lecture format for increasing resident interaction and knowledge achievement during noon conference: a prospective, controlled study.

    PubMed

    Sawatsky, Adam P; Berlacher, Kathryn; Granieri, Rosanne

    2014-07-01

    The traditional lecture is used by many residency programs to fulfill the mandate for regular didactic sessions, despite limited evidence to demonstrate its effectiveness. Active teaching strategies have shown promise in improving medical knowledge but have been challenging to implement within the constraints of residency training. We developed and evaluated an innovative structured format for interactive teaching within the residency noon conference. We developed an ACTIVE teaching format structured around the following steps: assemble (A) into groups, convey (C) learning objectives, teach (T) background information, inquire (I) through cases and questions, verify (V) understanding, and explain (E) answer choices and educate on the learning points. We conducted a prospective, controlled study of the ACTIVE teaching format versus the standard lecture format, comparing resident satisfaction, immediate knowledge achievement and long-term knowledge retention. We qualitatively assessed participating faculty members' perspectives on the faculty development efforts and the feasibility of teaching using the ACTIVE format. Sixty-nine internal medicine residents participated in the study. Overall, there was an improvement in perceived engagement using the ACTIVE teaching format (4.78 vs. 3.80, P < 0.01), with no increase in stress or decrement in break time. There was an improvement in initial knowledge achievement with the ACTIVE teaching format (overall absolute score increase of 11%, P = 0.04) and a trend toward improvement in long-term knowledge retention. Faculty members felt adequately prepared to use the ACTIVE teaching format, and enjoyed teaching with the ACTIVE teaching format more than the standard lecture. A structured ACTIVE teaching format improved resident engagement and initial knowledge, and required minimal resources. The ACTIVE teaching format offers an exciting alternative to the standard lecture for resident noon conference and is easy to implement.

  1. Using an ACTIVE teaching format versus a standard lecture format for increasing resident interaction and knowledge achievement during noon conference: a prospective, controlled study

    PubMed Central

    2014-01-01

    Background The traditional lecture is used by many residency programs to fulfill the mandate for regular didactic sessions, despite limited evidence to demonstrate its effectiveness. Active teaching strategies have shown promise in improving medical knowledge but have been challenging to implement within the constraints of residency training. We developed and evaluated an innovative structured format for interactive teaching within the residency noon conference. Methods We developed an ACTIVE teaching format structured around the following steps: assemble (A) into groups, convey (C) learning objectives, teach (T) background information, inquire (I) through cases and questions, verify (V) understanding, and explain (E) answer choices and educate on the learning points. We conducted a prospective, controlled study of the ACTIVE teaching format versus the standard lecture format, comparing resident satisfaction, immediate knowledge achievement and long-term knowledge retention. We qualitatively assessed participating faculty members’ perspectives on the faculty development efforts and the feasibility of teaching using the ACTIVE format. Results Sixty-nine internal medicine residents participated in the study. Overall, there was an improvement in perceived engagement using the ACTIVE teaching format (4.78 vs. 3.80, P < 0.01), with no increase in stress or decrement in break time. There was an improvement in initial knowledge achievement with the ACTIVE teaching format (overall absolute score increase of 11%, P = 0.04) and a trend toward improvement in long-term knowledge retention. Faculty members felt adequately prepared to use the ACTIVE teaching format, and enjoyed teaching with the ACTIVE teaching format more than the standard lecture. Conclusions A structured ACTIVE teaching format improved resident engagement and initial knowledge, and required minimal resources. The ACTIVE teaching format offers an exciting alternative to the standard lecture for

  2. Chapter 12: Daily Patterns of Marbled Murrelet Activity at Inland Sites

    Treesearch

    Nancy L. Naslund; Brian P. O’Donnell

    1995-01-01

    Patterns in the daily activity of Marbled Murrelets (Brachyramphus marmoratus) at inland sites has been studied throughout their range from California to Alaska. Murrelets are most active at inland sites around dawn, and to a lesser degree, at dusk. Throughout their range, peak levels of activity (detections) occur in the hour around dawn, but...

  3. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eren, Elif; Murphy, Megan; Goguen, Jon

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changesmore » of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.« less

  4. Combining active and passive seismic methods for the characterization of urban sites in Cairo, Egypt

    NASA Astrophysics Data System (ADS)

    Adly, Ashraf; Poggi, Valerio; Fäh, Donat; Hassoup, Awad; Omran, Awad

    2017-07-01

    The geology at Kottamiya, Rehab City and Zahraa-Madinat-Nasr to the East of Cairo (Egypt) is composed of low-velocity sediments on top of a rigid rock basement. Such sediments include the loose sands of the Gebel Ahmar formation, marl and shales of Maadi formation, in addition to sparse quaternary soil covers. Due to the contrast of the seismic impedance with the underlying bedrock, these soft sediments have the potential of considerably amplifying the ground motion during an earthquake. For the evaluation of site-specific seismic hazard, we computed the seismic site response in these areas by developing 1-D velocity models and derived average seismic velocities, including Vs30. To do that, we applied different active and passive source techniques, including the horizontal to vertical Fourier spectral ratio of ambient vibration recordings and multichannel analysis of artificially generated surface waves. A set of models representing the velocity structure of the site is then obtained by combined inversion of Rayleigh wave dispersion curves and ellipticity functions. While dispersion curves are used to constrain the uppermost low-velocity part of the soil profile, ellipticity helps in resolving the structure at the depth of the sediment-bedrock interface. From the retrieved velocity models, numerical ground-motion amplification is finally derived using 1-D SH-wave transfer function. We account for uncertainty in amplification by using a statistical model that accounts for the misfit of all the inverted velocity profiles. The study reveals that the different sites experience an important frequency-dependent amplification, with largest amplification occurring at the resonance frequencies of the sites. Amplification up to a factor of 5 is found, with some variability depending on the soil type (Vs30 ranges between 340 and 415 m s-2). Moreover, amplification is expected in the frequency range that is important for buildings (0.8-10 Hz), which is additional confirmation

  5. Dopant-Site Determination in Y- and Sc-Doped (Ba0.5Sr0.5)(Co0.8Fe0.2)O3-δ by Atom Location by Channeling Enhanced Microanalysis and the Role of Dopant Site on Secondary Phase Formation.

    PubMed

    Meffert, Matthias; Störmer, Heike; Gerthsen, Dagmar

    2016-02-01

    (Ba0.5Sr0.5)(Co0.8Fe0.2)O3-δ (BSCF) is a promising material with mixed ionic and electronic conductivity which is considered for oxygen separation membranes. Selective improvement of material properties, e.g. oxygen diffusivity or suppression of secondary phase formation, can be achieved by B-site doping. This study is concerned with the formation of Co-oxide precipitates in undoped BSCF at typical homogenization temperatures of 1,000°C, which act as undesirable nucleation sites for other secondary phases in the application-relevant temperature range. Y-doping successfully suppresses Co-oxide formation, whereas only minor improvements are achieved by Sc-doping. To understand the reason for the different behavior of Y and Sc, the lattice sites of dopant cations in BSCF were experimentally determined in this work. Energy-dispersive X-ray spectroscopy in a transmission electron microscope was applied to locate dopant sites exploiting the atom location by channeling enhanced microanalysis technique. It is shown that Sc exclusively occupies B-cation sites, whereas Y is detected on A- and B-cation sites in Y-doped BSCF, although solely B-site doping was intended. A model is presented for the suppression of Co-oxide formation in Y-doped BSCF based on Y double-site occupancy.

  6. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

  7. Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

    PubMed

    Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

    2014-10-01

    Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

  8. Active multilayered capsules for in vivo bone formation

    PubMed Central

    Facca, S.; Cortez, C.; Mendoza-Palomares, C.; Messadeq, N.; Dierich, A.; Johnston, A. P. R.; Mainard, D.; Voegel, J.-C.; Caruso, F.; Benkirane-Jessel, N.

    2010-01-01

    Interest in the development of new sources of transplantable materials for the treatment of injury or disease has led to the convergence of tissue engineering with stem cell technology. Bone and joint disorders are expected to benefit from this new technology because of the low self-regenerating capacity of bone matrix secreting cells. Herein, the differentiation of stem cells to bone cells using active multilayered capsules is presented. The capsules are composed of poly-L-glutamic acid and poly-L-lysine with active growth factors embedded into the multilayered film. The bone induction from these active capsules incubated with embryonic stem cells was demonstrated in vitro. Herein, we report the unique demonstration of a multilayered capsule-based delivery system for inducing bone formation in vivo. This strategy is an alternative approach for in vivo bone formation. Strategies using simple chemistry to control complex biological processes would be particularly powerful, as they make production of therapeutic materials simpler and more easily controlled. PMID:20160118

  9. Active-site solvent replenishment observed during human carbonic anhydrase II catalysis.

    PubMed

    Kim, Jin Kyun; Lomelino, Carrie L; Avvaru, Balendu Sankara; Mahon, Brian P; McKenna, Robert; Park, SangYoun; Kim, Chae Un

    2018-01-01

    Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO 2 /HCO 3 - . Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO 2 pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (W I ') is observed next to the previously observed intermediate water W I , and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH - on CO 2 . This mechanism explains how the zinc-bound water (W Zn ) and W1 are replenished, which are directly responsible for the reconnection of the His64-mediated proton-transfer water network. This study provides the first 'physical' glimpse of how a water reservoir flows into the hCA II active site during its catalytic activity.

  10. Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites.

    PubMed

    Ribeiro, António J M; Holliday, Gemma L; Furnham, Nicholas; Tyzack, Jonathan D; Ferris, Katherine; Thornton, Janet M

    2018-01-04

    M-CSA (Mechanism and Catalytic Site Atlas) is a database of enzyme active sites and reaction mechanisms that can be accessed at www.ebi.ac.uk/thornton-srv/m-csa. Our objectives with M-CSA are to provide an open data resource for the community to browse known enzyme reaction mechanisms and catalytic sites, and to use the dataset to understand enzyme function and evolution. M-CSA results from the merging of two existing databases, MACiE (Mechanism, Annotation and Classification in Enzymes), a database of enzyme mechanisms, and CSA (Catalytic Site Atlas), a database of catalytic sites of enzymes. We are releasing M-CSA as a new website and underlying database architecture. At the moment, M-CSA contains 961 entries, 423 of these with detailed mechanism information, and 538 with information on the catalytic site residues only. In total, these cover 81% (195/241) of third level EC numbers with a PDB structure, and 30% (840/2793) of fourth level EC numbers with a PDB structure, out of 6028 in total. By searching for close homologues, we are able to extend M-CSA coverage of PDB and UniProtKB to 51 993 structures and to over five million sequences, respectively, of which about 40% and 30% have a conserved active site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

    PubMed

    Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

    2018-05-03

    The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

  12. STAR FORMATION ACTIVITY IN CLASH BRIGHTEST CLUSTER GALAXIES

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fogarty, Kevin; Postman, Marc; Connor, Thomas

    2015-11-10

    The CLASH X-ray selected sample of 20 galaxy clusters contains 10 brightest cluster galaxies (BCGs) that exhibit significant (>5σ) extinction-corrected star formation rates (SFRs). Star formation activity is inferred from photometric estimates of UV and Hα+[N ii] emission in knots and filaments detected in CLASH Hubble Space Telescope ACS and WFC3 observations. UV-derived SFRs in these BCGs span two orders of magnitude, including two with a SFR ≳ 100 M{sub ⊙} yr{sup −1}. These measurements are supplemented with [O ii], [O iii], and Hβ fluxes measured from spectra obtained with the SOAR telescope. We confirm that photoionization from ongoing starmore » formation powers the line emission nebulae in these BCGs, although in many BCGs there is also evidence of a LINER-like contribution to the line emission. Coupling these data with Chandra X-ray measurements, we infer that the star formation occurs exclusively in low-entropy cluster cores and exhibits a correlation with gas properties related to cooling. We also perform an in-depth study of the starburst history of the BCG in the cluster RXJ1532.9+3021, and create 2D maps of stellar properties on scales down to ∼350 pc. These maps reveal evidence for an ongoing burst occurring in elongated filaments, generally on ∼0.5–1.0 Gyr timescales, although some filaments are consistent with much younger (≲100 Myr) burst timescales and may be correlated with recent activity from the active galactic nucleus. The relationship between BCG SFRs and the surrounding intracluster medium gas properties provide new support for the process of feedback-regulated cooling in galaxy clusters and is consistent with recent theoretical predictions.« less

  13. SABER: A computational method for identifying active sites for new reactions

    PubMed Central

    Nosrati, Geoffrey R; Houk, K N

    2012-01-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were l-Ala d/l-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

  14. SABER: a computational method for identifying active sites for new reactions.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. Copyright © 2012 The Protein Society.

  15. The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate

    PubMed Central

    Song, Guodong; Habibovic, Pamela; Bao, Chongyun; Hu, Jing; van Blitterswijk, Clemens A.; Yuan, Huipin; Chen, Wenchuan; Xu, Hockin H.K.

    2013-01-01

    Osteoinductive biomaterials are promising for bone repair. There is no direct proof that bone marrow mesenchymal stem cells (BMSCs) home to non-osseous sites and participate in ectopic bone formation induced by osteoinductive bioceramics. The objective of this study was to use a sex-mismatched beagle dog model to investigate BMSC homing via blood circulation to participate in ectopic bone formation via osteoinductive biomaterial. BMSCs of male dogs were injected into female femoral marrow cavity. The survival and stable chimerism of donor BMSCs in recipients were confirmed with polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Biphasic calcium phosphate (BCP) granules were implanted in dorsal muscles of female dogs. Y chromosomes were detected in samples harvested from female dogs which had received male BMSCs. At 4 weeks, cells with Y-chromosomes were distributed in the new bone matrix throughout the BCP granule implant. At 6 weeks, cells with Y chromosomes were present in newly mineralized woven bone. TRAP positive osteoclast-like cells were observed in 4-week implants, and the number of such cells decreased from 4 to 6 weeks. These results show that osteoprogenitors were recruited from bone marrow and homed to ectopic site to serve as a cell source for calcium phosphate-induced bone formation. In conclusion, BMSCs were demonstrated to migrate from bone marrow through blood circulation to non-osseous bioceramic implant site to contribute to ectopic bone formation in a canine model. BCP induced new bone in muscles without growth factor delivery, showing excellent osteoinductivity that could be useful for bone tissue engineering. PMID:23298780

  16. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-09

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

  17. Enhanced enzyme kinetic stability by increasing rigidity within the active site.

    PubMed

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-03-14

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

  18. GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

    PubMed

    Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

    2017-07-03

    Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Dioxygen Activation and O–O Bond Formation Reactions by Manganese Corroles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guo, Mian; Lee, Yong-Min; Gupta, Ranjana

    Activation of dioxygen (O 2) in enzymatic and biomimetic reactions has been intensively investigated over the past several decades. More recently, O–O bond formation, which is the reverse of the O 2-activation reaction, has been the focus of current research. Herein, we report the O 2-activation and O–O bond formation reactions by manganese corrole complexes. In the O 2-activation reaction, Mn(V)-oxo and Mn(IV)-peroxo intermediates were formed when Mn(III) corroles were exposed to O 2 in the presence of base (e.g., OH –) and hydrogen atom (H atom) donor (e.g., THF or cyclic olefins); the O 2-activation reaction did not occurmore » in the absence of base and H atom donor. Moreover, formation of the Mn(V)-oxo and Mn(IV)-peroxo species was dependent on the amounts of base present in the reaction solution. The role of the base was proposed to lower the oxidation potential of the Mn(III) corroles, thereby facilitating the binding of O 2 and forming a Mn(IV)-superoxo species. The putative Mn(IV)-superoxo species was then converted to the corresponding Mn(IV)-hydroperoxo species by abstracting a H atom from H atom donor, followed by the O–O bond cleavage of the putative Mn(IV)-hydroperoxo species to form a Mn(V)-oxo species. We have also shown that addition of hydroxide ion to the Mn(V)-oxo species afforded the Mn(IV)-peroxo species via O–O bond formation and the resulting Mn(IV)-peroxo species reverted to the Mn(V)-oxo species upon addition of proton, indicating that the O–O bond formation and cleavage reactions between the Mn(V)-oxo and Mn(IV)-peroxo complexes are reversible. The present paper reports the first example of using the same manganese complex in both O 2-activation and O–O bond formation reactions.« less

  20. Dioxygen Activation and O–O Bond Formation Reactions by Manganese Corroles

    DOE PAGES

    Guo, Mian; Lee, Yong-Min; Gupta, Ranjana; ...

    2017-10-22

    Activation of dioxygen (O 2) in enzymatic and biomimetic reactions has been intensively investigated over the past several decades. More recently, O–O bond formation, which is the reverse of the O 2-activation reaction, has been the focus of current research. Herein, we report the O 2-activation and O–O bond formation reactions by manganese corrole complexes. In the O 2-activation reaction, Mn(V)-oxo and Mn(IV)-peroxo intermediates were formed when Mn(III) corroles were exposed to O 2 in the presence of base (e.g., OH –) and hydrogen atom (H atom) donor (e.g., THF or cyclic olefins); the O 2-activation reaction did not occurmore » in the absence of base and H atom donor. Moreover, formation of the Mn(V)-oxo and Mn(IV)-peroxo species was dependent on the amounts of base present in the reaction solution. The role of the base was proposed to lower the oxidation potential of the Mn(III) corroles, thereby facilitating the binding of O 2 and forming a Mn(IV)-superoxo species. The putative Mn(IV)-superoxo species was then converted to the corresponding Mn(IV)-hydroperoxo species by abstracting a H atom from H atom donor, followed by the O–O bond cleavage of the putative Mn(IV)-hydroperoxo species to form a Mn(V)-oxo species. We have also shown that addition of hydroxide ion to the Mn(V)-oxo species afforded the Mn(IV)-peroxo species via O–O bond formation and the resulting Mn(IV)-peroxo species reverted to the Mn(V)-oxo species upon addition of proton, indicating that the O–O bond formation and cleavage reactions between the Mn(V)-oxo and Mn(IV)-peroxo complexes are reversible. The present paper reports the first example of using the same manganese complex in both O 2-activation and O–O bond formation reactions.« less

  1. A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

    PubMed

    Kudalkar, Shalley N; Njuma, Olive J; Li, Yongjiang; Muldowney, Michelle; Fuanta, N Rene; Goodwin, Douglas C

    2015-03-03

    Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

  2. Observations of fine particulate nitrated phenols in four sites in northern China: concentrations, source apportionment, and secondary formation

    NASA Astrophysics Data System (ADS)

    Wang, Liwei; Wang, Xinfeng; Gu, Rongrong; Wang, Hao; Yao, Lan; Wen, Liang; Zhu, Fanping; Wang, Weihao; Xue, Likun; Yang, Lingxiao; Lu, Keding; Chen, Jianmin; Wang, Tao; Zhang, Yuanghang; Wang, Wenxing

    2018-03-01

    Filter samples of fine particulate matters were collected at four sites in northern China (urban, rural, and mountain) in summer and winter, and the contents of nine nitrated phenols were quantified in the laboratory with the use of ultra-high-performance liquid chromatography coupled with mass spectrometry. During the sampling periods, the concentrations of particulate nitrated phenols exhibited distinct temporal and spatial variation. On average, the total concentration of particulate nitrated phenols in urban Jinan in the wintertime reached 48.4 ng m-3, and those in the summertime were 9.8, 5.7, 5.9, and 2.5 ng m-3 in urban Jinan, rural Yucheng and Wangdu, and Mt. Tai, respectively. The elevated concentrations of nitrated phenols in wintertime and in urban areas demonstrate the apparent influences of anthropogenic sources. The positive matrix factorization receptor model was then applied to determine the origins of particulate nitrated phenols in northern China. The five major source factors were traffic, coal combustion, biomass burning, secondary formation, and aged coal combustion plume. Among them, coal combustion played a vital role, especially at the urban site in the wintertime, with a contribution of around 55 %. In the summertime, the observed nitrated phenols were highly influenced by aged coal combustion plumes at all of the sampling sites. Meanwhile, in remote areas, contributions from secondary formation were significant. Further correlation analysis indicates that nitrosalicylic acids were produced mostly from secondary formation that was dominated by NO2 nitration.

  3. The Gαo Activator Mastoparan-7 Promotes Dendritic Spine Formation in Hippocampal Neurons.

    PubMed

    Ramírez, Valerie T; Ramos-Fernández, Eva; Inestrosa, Nibaldo C

    2016-01-01

    Mastoparan-7 (Mas-7), an analogue of the peptide mastoparan, which is derived from wasp venom, is a direct activator of Pertussis toxin- (PTX-) sensitive G proteins. Mas-7 produces several biological effects in different cell types; however, little is known about how Mas-7 influences mature hippocampal neurons. We examined the specific role of Mas-7 in the development of dendritic spines, the sites of excitatory synaptic contact that are crucial for synaptic plasticity. We report here that exposure of hippocampal neurons to a low dose of Mas-7 increases dendritic spine density and spine head width in a time-dependent manner. Additionally, Mas-7 enhances postsynaptic density protein-95 (PSD-95) clustering in neurites and activates Gα(o) signaling, increasing the intracellular Ca(2+) concentration. To define the role of signaling intermediates, we measured the levels of phosphorylated protein kinase C (PKC), c-Jun N-terminal kinase (JNK), and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) after Mas-7 treatment and determined that CaMKII activation is necessary for the Mas-7-dependent increase in dendritic spine density. Our results demonstrate a critical role for Gα(o) subunit signaling in the regulation of synapse formation.

  4. Ice Lens Formation and Frost Heave at the Phoenix Landing Site

    NASA Technical Reports Server (NTRS)

    Zent, A. P.; Sizemore, H. G.; Remple, A. W.

    2011-01-01

    Several lines of evidence indicate that the volume of shallow ground ice in the martian high latitudes exceeds the pore volume of the host regolith. Boynton et al. found an optimal fit to the Mars Odyssey Gamma Ray Spectrometer (GRS) data at the Phoenix landing site by modeling a buried layer of 50-75% ice by mass (up to 90% ice by volume). Thermal and optical observations of recent impact craters in the northern hemisphere have revealed nearly pure ice. Ice deposits containing only 1-2% soil by volume were excavated by Phoenix. The leading hypothesis for the origin of this excess ice is that it developed in situ by a mechanism analogous to the formation of terrestrial ice lenses and needle ice. Problematically, terrestrial soil-ice segregation is driven by freeze/thaw cycling and the movement of bulk water, neither of which are expected to have occurred in the geologically recent past on Mars. If however ice lens formation is possible at temperatures less than 273 K, there are possible implications for the habitability of Mars permafrost, since the same thin films of unfrozen water that lead to ice segregation are used by terrestrial psychrophiles to metabolize and grow down to temperatures of at least 258 K.

  5. Exploring the Dust Content, Metallicity, Star Formation and AGN Activity in Distant Dusty, Star-Forming Galaxies Using Cosmic Telescope

    NASA Astrophysics Data System (ADS)

    Walth, Gregory; Egami, Eiichi; Clément, Benjamin; Rujopakarn, Wiphu; Rawle, Tim; Richard, Johan; Dessauges, Miroslava; Perez-Gonzalez, Pablo; Ebeling, Harald; Vayner, Andrey; Wright, Shelley; Cosens, Maren; Herschel Lensing Survey

    2018-01-01

    We present our recent ALMA observations of Herschel-detected gravitationally lensed dusty, star-forming galaxies (DSFGs) and how they compliment our near-infrared spectroscopic observations of their rest-frame optical nebular emission. This provides the complete picture of star formation; from the molecular gas that fuels star formation, to the dust emission which are the sites of star formation, and the nebular emission which is the gas excited by the young stars. DSFGs undergo the largest starbursts in the Universe, contributing to the bulk of the cosmic star formation rate density between redshifts z = 1 - 4. Internal processes within high-redshift DSFGs remains largely unexplored; such as feedback from star formation, the role of turbulence, gas surface density of molecular gas, AGN activity, and the rates of metal production. Much that is known about DSFGs star formation properties comes from their CO and dust emission. In order to fully understand the star formation history of DSFGs, it is necessary to observe their optical nebular emission. Unfortunately, UV/optical emission is severely attenuated by dust, making it challenging to detect. With the Herschel Lensing Survey, a survey of the cores of almost 600 massive galaxy clusters, we are able to probe faint dust-attenuated nebular emission. We are currently conducting a new survey using Keck/OSIRIS to resolve a sample of gravitationally lensed DSFGs from the Herschel Lensing Survey (>100 mJy, with SFRs >100 Msun/yr) at redshifts z=1-4 with magnifications >10x all with previously detected nebular emission lines. We present the physical and resolved properties of gravitationally lensed DSFGs at unprecedented spatial scales; such as ionization, metallicity, AGN activity, and dust attenuation.

  6. Facebook, Twitter Activities Sites, Location and Students' Interest in Learning

    ERIC Educational Resources Information Center

    Igbo, J. N.; Ezenwaji, Ifeyinwa; Ajuziogu, Christiana U.

    2018-01-01

    This study was carried out to ascertain the influence of social networking sites activities (twitter and Facebook) on secondary school students' interest in learning It also considered the impact of these social networking sites activities on location of the students. Two research questions and two null hypotheses guided the study. Mean and…

  7. Comparing the validity of 2 physical activity questionnaire formats in African-American and Hispanic women.

    PubMed

    Mâsse, Louise C; Fulton, Janet E; Watson, Kathleen B; Tortolero, Susan; Kohl, Harold W; Meyers, Michael C; Blair, Steven N; Wong, William W

    2012-02-01

    The purpose of this study was to compare the validity of 2 physical activity questionnaire formats--one that lists activities (Checklist questionnaire) and one that assesses overall activities (Global questionnaire) by domain. Two questionnaire formats were validated among 260 African-American and Hispanic women (age 40-70) using 3 validation standards: 1) accelerometers to validate activities of ambulation; 2) diaries to validate physical activity domains (occupation, household, exercise, yard, family, volunteer/church work, and transportation); and 3) doubly-labeled water to validate physical activity energy expenditure (DLW-PAEE). The proportion of total variance explained by the Checklist questionnaire was 38.4% with diaries, 9.0% with accelerometers, and 6.4% with DLW-PAEE. The Global questionnaire explained 17.6% of the total variance with diaries and about 5% with both accelerometers and with DLWPAEE. Overall, associations with the 3 validation standards were slightly better with the Checklist questionnaire. However, agreement with DLW-PAEE was poor with both formats and the Checklist format resulted in greater overestimation. Validity results also indicated the Checklist format was better suited to recall household, family, and transportation activities. Overall, the Checklist format had slightly better measurement properties than the Global format. Both questionnaire formats are better suited to rank individuals.

  8. Different contributions of primary motor cortex, reticular formation, and spinal cord to fractionated muscle activation.

    PubMed

    Zaaimi, Boubker; Dean, Lauren R; Baker, Stuart N

    2018-01-01

    Coordinated movement requires patterned activation of muscles. In this study, we examined differences in selective activation of primate upper limb muscles by cortical and subcortical regions. Five macaque monkeys were trained to perform a reach and grasp task, and electromyogram (EMG) was recorded from 10 to 24 muscles while weak single-pulse stimuli were delivered through microelectrodes inserted in the motor cortex (M1), reticular formation (RF), or cervical spinal cord (SC). Stimulus intensity was adjusted to a level just above threshold. Stimulus-evoked effects were assessed from averages of rectified EMG. M1, RF, and SC activated 1.5 ± 0.9, 1.9 ± 0.8, and 2.5 ± 1.6 muscles per site (means ± SD); only M1 and SC differed significantly. In between recording sessions, natural muscle activity in the home cage was recorded using a miniature data logger. A novel analysis assessed how well natural activity could be reconstructed by stimulus-evoked responses. This provided two measures: normalized vector length L, reflecting how closely aligned natural and stimulus-evoked activity were, and normalized residual R, measuring the fraction of natural activity not reachable using stimulus-evoked patterns. Average values for M1, RF, and SC were L = 119.1 ± 9.6, 105.9 ± 6.2, and 109.3 ± 8.4% and R = 50.3 ± 4.9, 56.4 ± 3.5, and 51.5 ± 4.8%, respectively. RF was significantly different from M1 and SC on both measurements. RF is thus able to generate an approximation to the motor output with less activation than required by M1 and SC, but M1 and SC are more precise in reaching the exact activation pattern required. Cortical, brainstem, and spinal centers likely play distinct roles, as they cooperate to generate voluntary movements. NEW & NOTEWORTHY Brainstem reticular formation, primary motor cortex, and cervical spinal cord intermediate zone can all activate primate upper limb muscles. However, brainstem output is more

  9. Different contributions of primary motor cortex, reticular formation, and spinal cord to fractionated muscle activation

    PubMed Central

    Dean, Lauren R.

    2018-01-01

    Coordinated movement requires patterned activation of muscles. In this study, we examined differences in selective activation of primate upper limb muscles by cortical and subcortical regions. Five macaque monkeys were trained to perform a reach and grasp task, and electromyogram (EMG) was recorded from 10 to 24 muscles while weak single-pulse stimuli were delivered through microelectrodes inserted in the motor cortex (M1), reticular formation (RF), or cervical spinal cord (SC). Stimulus intensity was adjusted to a level just above threshold. Stimulus-evoked effects were assessed from averages of rectified EMG. M1, RF, and SC activated 1.5 ± 0.9, 1.9 ± 0.8, and 2.5 ± 1.6 muscles per site (means ± SD); only M1 and SC differed significantly. In between recording sessions, natural muscle activity in the home cage was recorded using a miniature data logger. A novel analysis assessed how well natural activity could be reconstructed by stimulus-evoked responses. This provided two measures: normalized vector length L, reflecting how closely aligned natural and stimulus-evoked activity were, and normalized residual R, measuring the fraction of natural activity not reachable using stimulus-evoked patterns. Average values for M1, RF, and SC were L = 119.1 ± 9.6, 105.9 ± 6.2, and 109.3 ± 8.4% and R = 50.3 ± 4.9, 56.4 ± 3.5, and 51.5 ± 4.8%, respectively. RF was significantly different from M1 and SC on both measurements. RF is thus able to generate an approximation to the motor output with less activation than required by M1 and SC, but M1 and SC are more precise in reaching the exact activation pattern required. Cortical, brainstem, and spinal centers likely play distinct roles, as they cooperate to generate voluntary movements. NEW & NOTEWORTHY Brainstem reticular formation, primary motor cortex, and cervical spinal cord intermediate zone can all activate primate upper limb muscles. However, brainstem output is more

  10. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  11. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

    PubMed

    Blaha-Nelson, David; Krüger, Dennis M; Szeler, Klaudia; Ben-David, Moshe; Kamerlin, Shina Caroline Lynn

    2017-01-25

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the k cat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.

  12. Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

    PubMed

    Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

    2016-11-01

    Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

  13. STAR FORMATION ACTIVITY IN THE GALACTIC H II COMPLEX S255-S257

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ojha, D. K.; Ghosh, S. K.; Samal, M. R.

    We present results on the star formation activity of an optically obscured region containing an embedded cluster (S255-IR) and molecular gas between two evolved H II regions, S255 and S257. We have studied the complex using optical and near-infrared (NIR) imaging, optical spectroscopy, and radio continuum mapping at 15 GHz, along with Spitzer-IRAC results. We found that the main exciting sources of the evolved H II regions S255 and S257 and the compact H II regions associated with S255-IR are of O9.5-B3 V nature, consistent with previous observations. Our NIR observations reveal 109 likely young stellar object (YSO) candidates inmore » an area of {approx}4.'9 x 4.'9 centered on S255-IR, which include 69 new YSO candidates. To see the global star formation, we constructed the V - I/V diagram for 51 optically identified IRAC YSOs in an area of {approx}13' x 13' centered on S255-IR. We suggest that these YSOs have an approximate age between 0.1 and 4 Myr, indicating a non-coeval star formation. Using spectral energy distribution models, we constrained physical properties and evolutionary status of 31 and 16 YSO candidates outside and inside the gas ridge, respectively. The models suggest that the sources associated with the gas ridge are younger (mean age {approx}1.2 Myr) than the sources outside the gas ridge (mean age {approx}2.5 Myr). The positions of the young sources inside the gas ridge at the interface of the H II regions S255 and S257 favor a site of induced star formation.« less

  14. The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gopal, B.; Madan, Lalima L.; Betz, Stephen F.

    2010-11-10

    Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s),more » whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.« less

  15. Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

    PubMed Central

    Hennig, Sandra E.; Jeoung, Jae-Hun; Goetzl, Sebastian; Dobbek, Holger

    2012-01-01

    Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe4S4] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins. PMID:22431597

  16. Ligand versus metal protonation of an iron hydrogenase active site mimic.

    PubMed

    Eilers, Gerriet; Schwartz, Lennart; Stein, Matthias; Zampella, Giuseppe; de Gioia, Luca; Ott, Sascha; Lomoth, Reiner

    2007-01-01

    The protonation behavior of the iron hydrogenase active-site mimic [Fe2(mu-adt)(CO)4(PMe3)2] (1; adt=N-benzyl-azadithiolate) has been investigated by spectroscopic, electrochemical, and computational methods. The combination of an adt bridge and electron-donating phosphine ligands allows protonation of either the adt nitrogen to give [Fe2(mu-Hadt)(CO)4(PMe3)2]+ ([1 H]+), the Fe-Fe bond to give [Fe2(mu-adt)(mu-H)(CO)4(PMe3)2]+ ([1 Hy]+), or both sites simultaneously to give [Fe2(mu-Hadt)(mu-H)(CO)4(PMe3)2]2+ ([1 HHy]2 +). Complex 1 and its protonation products have been characterized in acetonitrile solution by IR, (1)H, and (31)P NMR spectroscopy. The solution structures of all protonation states feature a basal/basal orientation of the phosphine ligands, which contrasts with the basal/apical structure of 1 in the solid state. Density functional calculations have been performed on all protonation states and a comparison between calculated and experimental spectra confirms the structural assignments. The ligand protonated complex [1 H]+ (pKa=12) is the initial, metastable protonation product while the hydride [1 Hy]+ (pKa=15) is the thermodynamically stable singly protonated form. Tautomerization of cation [1 H]+ to [1 Hy]+ does not occur spontaneously. However, it can be catalyzed by HCl (k=2.2 m(-1) s(-1)), which results in the selective formation of cation [1 Hy]+. The protonations of the two basic sites have strong mutual effects on their basicities such that the hydride (pK(a)=8) and the ammonium proton (pK(a)=5) of the doubly protonated cationic complex [1 HHy]2+ are considerably more acidic than in the singly protonated analogues. The formation of dication [1 HHy]2+ from cation [1 H]+ is exceptionally slow with perchloric or trifluoromethanesulfonic acid (k=0.15 m(-1) s(-1)), while the dication is formed substantially faster (k>10(2) m(-1) s(-1)) with hydrobromic acid. Electrochemically, 1 undergoes irreversible reduction at -2.2 V versus ferrocene, and this

  17. 12 CFR Appendix I to Part 27 - Monthly Home Loan Activity Format

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 1 2011-01-01 2011-01-01 false Monthly Home Loan Activity Format I Appendix I to Part 27 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY FAIR HOUSING HOME LOAN DATA SYSTEM Pt. 27, App. I Appendix I to Part 27—Monthly Home Loan Activity Format EC22SE91...

  18. 12 CFR Appendix I to Part 27 - Monthly Home Loan Activity Format

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 1 2013-01-01 2013-01-01 false Monthly Home Loan Activity Format I Appendix I to Part 27 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY FAIR HOUSING HOME LOAN DATA SYSTEM Pt. 27, App. I Appendix I to Part 27—Monthly Home Loan Activity Format EC22SE91...

  19. 12 CFR Appendix I to Part 27 - Monthly Home Loan Activity Format

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Monthly Home Loan Activity Format I Appendix I to Part 27 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY FAIR HOUSING HOME LOAN DATA SYSTEM Pt. 27, App. I Appendix I to Part 27—Monthly Home Loan Activity Format EC22SE91...

  20. 12 CFR Appendix I to Part 27 - Monthly Home Loan Activity Format

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 1 2012-01-01 2012-01-01 false Monthly Home Loan Activity Format I Appendix I to Part 27 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY FAIR HOUSING HOME LOAN DATA SYSTEM Pt. 27, App. I Appendix I to Part 27—Monthly Home Loan Activity Format EC22SE91...

  1. 12 CFR Appendix I to Part 27 - Monthly Home Loan Activity Format

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Monthly Home Loan Activity Format I Appendix I to Part 27 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY FAIR HOUSING HOME LOAN DATA SYSTEM Pt. 27, App. I Appendix I to Part 27—Monthly Home Loan Activity Format EC22SE91...

  2. Xanthine Oxidase Induces Foam Cell Formation through LOX-1 and NLRP3 Activation.

    PubMed

    Dai, Yao; Cao, Yongxiang; Zhang, Zhigao; Vallurupalli, Srikanth; Mehta, Jawahar L

    2017-02-01

    Xanthine oxidase catalyzes the oxidation of xanthine to uric acid. This process generates excessive reactive oxygen species (ROS) that play an important role in atherogenesis. Recent studies show that LRR and PYD domains-containing protein 3 (NLRP3), a component of the inflammasome, may be involved in the formation of foam cells, a hallmark of atherosclerosis. This study was designed to study the role of various scavenger receptors and NLRP3 inflammasome in xanthine oxidase and uric acid-induced foam cell formation. Human vascular smooth muscle cells (VSMCs) and THP-1 macrophages were treated with xanthine oxidase or uric acid. Xanthine oxidase treatment (of both VSMCs and THP-1 cells) resulted in foam cell formation in concert with generation of ROS and expression of cluster of differentiation 36 (CD36) and oxidized low density lipoprotein (lectin-like) receptor 1 (LOX-1), but not of scavenger receptor A (SRA). Uric acid treatment resulted in foam cell formation, ROS generation and expression of CD36, but not of LOX-1 or SRA. Further, treatment of cells with xanthine oxidase, but not uric acid, activated NLRP3 and its downstream pro-inflammatory signals- caspase-1, interleukin (IL)-1β and IL-18. Blockade of LOX-1 or NLRP3 inflammasome with specific siRNAs reduced xanthine oxidase-induced foam cell formation, ROS generation and activation of NLRP3 and downstream signals. Xanthine oxidase induces foam cell formation in large part through activation of LOX-1 - NLRP3 pathway in both VSMCs and THP-1 cells, but uric acid-induced foam cell formation is exclusively through CD36 pathway. Further, LOX-1 activation is upstream of NLRP3 activation. Graphical Abstract Steps in the formation of foam cells in response to xanthine oxidase and uric acid. Xanthine oxidase stimulates LOX-1 expression on the cell membrane of macrophages and vascular smooth muscle cells (VSMCs) and increases generation of ROS, which activate NLRP3 inflammasome and downstream pro

  3. A hybrid QM/MM simulation study of intramolecular proton transfer in the pyridoxal 5'-phosphate in the active site of transaminase: influence of active site interaction on proton transfer.

    PubMed

    Dutta Banik, Sindrila; Chandra, Amalendu

    2014-09-25

    Pyridoxal 5'-phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium that involves an intramolecular proton transfer between the N-protonated zwitterionic ketoenamine tautomer and the O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in the zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP aspartate (PLP-Asp) Schiff base in the active site of aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical simulations. The main focus of the present study is to analyze the factors that control the tautomeric equilibrium in the active sites of various PLP-dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for the ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in the ketoenamine tautomer, while in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP-dependent enzymes (aspartate aminotransferase, Dopa decarboxylase, and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium toward the N

  4. Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

    PubMed Central

    Ganter, U M; Longstaff, C; Pajares, M A; Rando, R R; Siebert, F

    1991-01-01

    Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species. PMID:2049524

  5. Star Formation Activity in CLASH Brightest Cluster Galaxies

    NASA Astrophysics Data System (ADS)

    Fogarty, Kevin; Postman, Marc; Connor, Thomas; Donahue, Megan; Moustakas, John

    2015-11-01

    The CLASH X-ray selected sample of 20 galaxy clusters contains 10 brightest cluster galaxies (BCGs) that exhibit significant (>5σ) extinction-corrected star formation rates (SFRs). Star formation activity is inferred from photometric estimates of UV and Hα+[N ii] emission in knots and filaments detected in CLASH Hubble Space Telescope ACS and WFC3 observations. UV-derived SFRs in these BCGs span two orders of magnitude, including two with a SFR ≳ 100 M⊙ yr-1. These measurements are supplemented with [O ii], [O iii], and Hβ fluxes measured from spectra obtained with the SOAR telescope. We confirm that photoionization from ongoing star formation powers the line emission nebulae in these BCGs, although in many BCGs there is also evidence of a LINER-like contribution to the line emission. Coupling these data with Chandra X-ray measurements, we infer that the star formation occurs exclusively in low-entropy cluster cores and exhibits a correlation with gas properties related to cooling. We also perform an in-depth study of the starburst history of the BCG in the cluster RXJ1532.9+3021, and create 2D maps of stellar properties on scales down to ˜350 pc. These maps reveal evidence for an ongoing burst occurring in elongated filaments, generally on ˜0.5-1.0 Gyr timescales, although some filaments are consistent with much younger (≲100 Myr) burst timescales and may be correlated with recent activity from the active galactic nucleus. The relationship between BCG SFRs and the surrounding intracluster medium gas properties provide new support for the process of feedback-regulated cooling in galaxy clusters and is consistent with recent theoretical predictions. Based on observations obtained at the Southern Astrophysical Research (SOAR) telescope, which is a joint project of the Ministério da Ciência, Tecnologia, e Inovação (MCTI) da República Federativa do Brasil, the U.S. National Optical Astronomy Observatory (NOAO), the University of North Carolina at Chapel

  6. Leptin promotes neointima formation and smooth muscle cell proliferation via NADPH oxidase activation and signalling in caveolin-rich microdomains.

    PubMed

    Schroeter, Marco R; Leifheit-Nestler, Maren; Hubert, Astrid; Schumann, Bettina; Glückermann, Roland; Eschholz, Norman; Krüger, Nenja; Lutz, Susanne; Hasenfuss, Gerd; Konstantinides, Stavros; Schäfer, Katrin

    2013-08-01

    Apolipoprotein E (apoE) may act as a vasculoprotective factor by promoting plasma lipid clearance and cholesterol efflux. Moreover, apoE accumulates at sites of vascular injury and modulates the effect of growth factors on smooth muscle cells (SMCs). Experimental data suggested that hypothalamic apoE expression is reduced in obesity and associated with leptin resistance. In this study, we examined the role of apoE in mediating the effects of leptin on vascular lesion formation. Leptin was administered to apoE knockout (apoE-/-) mice via osmotic pumps to increase its circulating levels. Morphometric analysis revealed that leptin did not alter neointima formation and failed to increase α-actin- or PCNA-immunopositive SMCs after vascular injury. Similar findings were obtained after analysis of atherosclerotic lesions. Comparison of apoE-/-, wild-type, or LDL receptor-/- mice and functional analyses in aortic SMCs from WT or apoE-/- mice or human arterial SMCs after treatment with small interfering (si)RNA or heparinase revealed that leptin requires the presence of apoE, expressed, secreted and bound to the cell surface, to fully activate leptin receptor signalling and to promote SMC proliferation and neointima formation. Mechanistically, leptin induced the phosphorylation and membrane translocation of caveolin (cav)-1, and apoE down-regulation or caveolae disruption inhibited the leptin-induced p47phox activation, ROS formation and SMC proliferation. Finally, leptin failed to increase neointima formation in mice lacking cav-1. Our findings suggest that apoE mediates the effects of leptin on vascular lesion formation by stabilizing cav-1-enriched cell membrane microdomains in SMCs, thus allowing NADPH oxidase assembly and ROS-mediated mitogenic signalling.

  7. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  8. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites.

    PubMed

    Chen, Baoyu; Chou, Hui-Ting; Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas; Rosen, Michael K

    2017-09-26

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

  9. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    DOE PAGES

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [Cu IIOH] + species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [Cu IIOH] + active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu] 2+ and Cu 3O 3 motifs.« less

  10. Flavonol Activation Defines an Unanticipated Ligand-Binding Site in the Kinase-RNase Domain of IRE1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wiseman, R. Luke; Zhang, Yuhong; Lee, Kenneth P.K.

    2010-08-18

    Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence ofmore » enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.« less

  11. Surface Formation and Preservation of Very-Low-Porosity Thin Crusts ( "Glazes") at the WAIS Divide Site, West Antarctica

    NASA Astrophysics Data System (ADS)

    Fegyveresi, J. M.; Alley, R. B.; Muto, A.; Spencer, M. K.; Orsi, A. J.

    2014-12-01

    Observations at the WAIS Divide site show that near-surface snow is strongly altered by weather-related processes, producing features that are recognizable in the ice core. Prominent reflective "glazed" surface crusts develop frequently during the summer. Observations during austral summers 2008-09 through 2012-13, supplemented by Automated Weather Station data with insolation sensors, documented formation of such crusts during relatively low-wind, low-humidity, clear-sky periods with intense daytime sunshine. After formation, such glazed surfaces typically developed cracks in a polygonal pattern with few-meter spacing, likely from thermal contraction at night. Cracking was commonest when several clear days occurred in succession, and was generally followed by surface hoar growth. Temperature and radiation observations showed that solar heating often warmed the near-surface snow above the air temperature, contributing to mass transfer favoring crust formation. Subsequent investigation of the WDC06A deep ice core revealed that preserved surface crusts were seen in the core at an average rate of ~4.3 ± 2 yr-1 over the past 5500 years. They are about 40% more common in layers deposited during summers than during winters. The total summertime crust frequency also covaried with site temperature, with more present during warmer periods. We hypothesize that the mechanism for glaze formation producing single-grain-thick very-low-porosity thin crusts (i.e. "glazes") involves additional in-filling of open pores. The thermal conductivity of ice greatly exceeds that of air, so heat transport in firn is primarily conductive. Because heat flow is primarily through the grain structure, for a temperature inversion (colder upper surface) beneath a growing thin crust at the upper surface, pores will be colder than interconnected grains, favoring mass transport into those pores. Transport may occur by vapor, surface, or volume diffusion, although vapor diffusion and surface

  12. Prostaglandin E2 regulates melanocyte dendrite formation through activation of PKCζ

    PubMed Central

    Scott, Glynis; Fricke, Alex; Fender, Anne; McClelland, Lindy; Jacobs, Stacey

    2007-01-01

    Prostaglandins are lipid signaling intermediates released by keratinocytes in response to ultraviolet irradiation (UVR) in the skin. The main prostaglandin released following UVR is PGE2, a ligand for 4 related G-protein coupled receptors (EP1, EP2, EP3 and EP4). Our previous work established that PGE2 stimulates melanocyte dendrite formation through activation of the EP1 and EP3 receptors. The purpose of the present report is to define the signaling intermediates involved in EP1 and EP3-dependent dendrite formation in human melanocytes. We recently showed that activation of the atypical PKCζ isoform stimulates melanocyte dendricity in response to treatment with lysophosphatidylcholine. We therefore examined the potential contribution of PKCζ activation on EP1 and EP3-dependent dendrite formation in melanocytes. Stimulation of the EP1 and EP3 receptors by selective agonists activated PKCζ, and inhibition of PKCζ activation abrogated EP1 and EP3-receptor mediated melanocyte dendricity. Because of the importance of Rho-GTP binding proteins in the regulation of melanocyte dendricity, we also examined the effect of EP1 and EP3 receptor activation on Rac and Rho activity. Neither Rac nor Rho was activated upon treatment with EP1,3-receptor agonists. We show that melanocytes express only the EP3A1 isoform, but not the EP3B receptor isoform, previously associated with Rho activation, consistent with a lack of Rho stimulation by EP3 agonists. Our data suggest that PKCζ activation plays a predominant role in regulation of PGE2-dependent melanocyte dendricity. PMID:17850789

  13. αMβ₂ integrin activation prevents alternative activation of human and murine macrophages and impedes foam cell formation.

    PubMed

    Yakubenko, Valentin P; Bhattacharjee, Ashish; Pluskota, Elzbieta; Cathcart, Martha K

    2011-03-04

    The alternative activation of monocytes by interleukin (IL)-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. In this report, we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13-mediated monocyte activation. We focused on the analysis of 2 proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis, an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. We found that adhesion of resting monocytes through β(2) integrins and inside-out activation of β(2) integrins by monocyte chemoattractant protein-1 did not change IL-13-stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of β(2) integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of β(1) integrins had no effect on 15-LO expression. Analysis of integrin clustering through α(M), α(L), α(X), and α(D) subunits demonstrated the pivotal role for integrin α(M)β(2) in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO-dependent CD36 expression on human monocytes; our studies showed that β(2) integrin activation and α(M) integrin clustering significantly inhibited IL-13-dependent CD36 mRNA and protein expression, as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of α(M)-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression, and lipid accumulation as compared with wild-type controls. The adhesion of monocytes/macrophages through activated integrin α(M)β(2) has a regulatory and potential atheroprotective function during the alternative activation of macrophages.

  14. αMβ2 Integrin Activation Prevents Alternative Activation of Human and Murine Macrophages and Impedes Foam Cell Formation

    PubMed Central

    Yakubenko, Valentin P.; Bhattacharjee, Ashish; Pluskota, Elzbieta; Cathcart, Martha K.

    2011-01-01

    Rationale The alternative activation of monocytes by IL-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. Objective In this paper we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13-mediated monocyte activation. We focused on the analysis of two proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis - an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. Methods and Results We found that adhesion of resting monocytes through β2 integrins and inside-out activation of β2 integrins by MCP-1 did not change IL-13-stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of β2 integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of β1 integrins had no effect on 15-LO expression. Analysis of integrin clustering through αM, αL, αX and αD subunits demonstrated the pivotal role for integrin αMβ2 in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO-dependent CD36 expression on human monocytes, our studies showed that β2 integrin activation and αM integrin clustering significantly inhibited IL-13-dependent CD36 mRNA and protein expression as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of αM-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression and lipid accumulation as compared to wild type controls. Conclusions The adhesion of monocytes/macrophages through activated integrin αMβ2 has a regulatory and potential athero-protective function during the alternative activation of macrophages. PMID:21252155

  15. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster.

  16. What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

    PubMed

    Zhang, Ni; Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

    2017-06-22

    Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. ©Ni Zhang, Shelly Campo, Jingzhen Yang, Petya Eckler, Linda Snetselaar, Kathleen Janz, Emily Leary. Originally published in the Journal of Medical

  17. What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

    PubMed Central

    Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

    2017-01-01

    Background Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. Objective This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. Methods A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. Results The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. Conclusions This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. PMID:28642215

  18. Effect of heparin bonding on catheter-induced fibrin formation and platelet activation.

    PubMed

    Nichols, A B; Owen, J; Grossman, B A; Marcella, J J; Fleisher, L N; Lee, M M

    1984-11-01

    Pathologic and experimental evidence indicates that platelet activation and fibrin formation contribute to the pathogenesis of angina pectoris, coronary vasospasm and myocardial infarction. Detection of localized intravascular platelet activation and fibrin formation in vivo by selective blood sampling requires catheters that do not induce coagulation ex vivo. We studied the effect of heparin bonding of catheter surfaces on activation of the coagulation system by cardiovascular catheters. Woven Dacron, polyvinylchloride, and polyurethane catheters were tested and compared with identical catheters with heparin-bonded surfaces in 47 patients undergoing percutaneous cardiac catheterization. Platelet activation was measured by radioimmunoassay of plasma platelet factor 4 (PF4), beta-thromboglobulin (BTG), and thromboxane B2 (TXB2) in blood samples withdrawn through catheters, and fibrin formation was assessed by determination of fibrinopeptide A (FPA) levels. In blood samples collected through conventional catheters, FPA, PF4, BTG, and TXB2 levels were markedly elevated; blood sampling through heparin-bonded catheters had no significant effect on FPA, PF4, BTG, or TXB2 levels. Scanning electron microscopy disclosed extensive platelet aggregates and fibrin strands adherent to the surface of conventional catheters but not to heparin-bonded catheter surfaces. This study demonstrates that (1) collection of blood samples through cardiovascular catheters causes artifactual elevation of FPA, PF4, BTG, and TXB2 levels, and (2) heparin-bonded catheter surfaces effectively prevent catheter-induced platelet alpha-granule release and fibrin formation on catheter surfaces. Heparin-bonded catheters will facilitate investigation of the role of intravascular coagulation in coronary artery disease by eliminating catheter-induced fibrin formation and platelet activation.

  19. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  20. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  1. Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics.

    PubMed

    Bauler, Patricia; Huber, Gary; Leyh, Thomas; McCammon, J Andrew

    2010-05-06

    Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.

  2. Disulfide Bond Formation and ToxR Activity in Vibrio cholerae

    PubMed Central

    Fengler, Vera H. I.; Boritsch, Eva C.; Tutz, Sarah; Seper, Andrea; Ebner, Hanna; Roier, Sandro; Schild, Stefan; Reidl, Joachim

    2012-01-01

    Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation. PMID:23144706

  3. The Gα o Activator Mastoparan-7 Promotes Dendritic Spine Formation in Hippocampal Neurons

    PubMed Central

    Ramírez, Valerie T.; Ramos-Fernández, Eva; Inestrosa, Nibaldo C.

    2016-01-01

    Mastoparan-7 (Mas-7), an analogue of the peptide mastoparan, which is derived from wasp venom, is a direct activator of Pertussis toxin- (PTX-) sensitive G proteins. Mas-7 produces several biological effects in different cell types; however, little is known about how Mas-7 influences mature hippocampal neurons. We examined the specific role of Mas-7 in the development of dendritic spines, the sites of excitatory synaptic contact that are crucial for synaptic plasticity. We report here that exposure of hippocampal neurons to a low dose of Mas-7 increases dendritic spine density and spine head width in a time-dependent manner. Additionally, Mas-7 enhances postsynaptic density protein-95 (PSD-95) clustering in neurites and activates Gα o signaling, increasing the intracellular Ca2+ concentration. To define the role of signaling intermediates, we measured the levels of phosphorylated protein kinase C (PKC), c-Jun N-terminal kinase (JNK), and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) after Mas-7 treatment and determined that CaMKII activation is necessary for the Mas-7-dependent increase in dendritic spine density. Our results demonstrate a critical role for Gα o subunit signaling in the regulation of synapse formation. PMID:26881110

  4. Revised description of index of Florida water data collection active stations and a user's guide for station or site information retrieval computer program FINDEX H578

    USGS Publications Warehouse

    Geiger, Linda H.

    1983-01-01

    The report is an update of U.S. Geological Survey Open-File Report 77-703, which described a retrieval program for administrative index of active data-collection sites in Florida. Extensive changes to the Findex system have been made since 1977 , making the previous report obsolete. A description of the data base and computer programs that are available in the Findex system are documented in this report. This system serves a vital need in the administration of the many and diverse water-data collection activities. District offices with extensive data-collection activities will benefit from the documentation of the system. Largely descriptive, the report tells how a file of computer card images has been established which contains entries for all sites in Florida at which there is currently a water-data collection activity. Entries include information such as identification number, station name, location, type of site, county, frequency of data collection, funding, and other pertinent details. The computer program FINDEX selectively retrieves entries and lists them in a format suitable for publication. The index is updated routinely. (USGS)

  5. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben

    2017-03-28

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by themore » SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.« less

  6. Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

    PubMed

    Farb, Joshua N; Morrical, Scott W

    2009-01-16

    Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

  7. Formation, Migration, and Reactivity of Au CO Complexes on Gold Surfaces

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Jun; McEntee, Monica; Tang, Wenjie

    2016-01-12

    Here, we report experimental as well as theoretical evidence that suggests Au CO complex formation upon the exposure of CO to active sites (step edges and threading dislocations) on a Au(111) surface. Room-temperature scanning tunneling microscopy (STM), X-ray photoelectron spectroscopy, transmission infrared spectroscopy, and density functional theory calculations point to Au CO complex formation and migration. Room-temperature STM of the Au(111) surface at CO pressures in the range from 10^ 8 to 10^ 4 Torr (dosage up to 10^6 langmuir) indicates Au atom extraction from dislocation sites of the herringbone reconstruction, mobile Au CO complex formation and diffusion, and Aumore » adatom cluster formation on both elbows and step edges on the Au surface. The formation and mobility of the Au CO complex result from the reduced Au Au bonding at elbows and step edges leading to stronger Au CO bonding and to the formation of a more positively charged CO (CO +) on Au. These studies indicate that the mobile Au CO complex is involved in the Au nanoparticle formation and reactivity, and that the positive charge on CO increases due to the stronger adsorption of CO at Au sites with lower coordination numbers.« less

  8. Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

    PubMed

    Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2018-02-14

    Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

  9. CASPASE-9 CARD:CORE DOMAIN INTERACTIONS REQUIRE A PROPERLY-FORMED ACTIVE SITE

    PubMed Central

    Huber, Kristen L.; Serrano, Banyuhay P.; Hardy, Jeanne A.

    2018-01-01

    Caspase-9 is a critical factor in the initiation of apoptosis, and as a result is tightly regulated by a number of mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Together these findings provide mechanistic insight into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate. PMID:29500231

  10. Prediction of active sites of enzymes by maximum relevance minimum redundancy (mRMR) feature selection.

    PubMed

    Gao, Yu-Fei; Li, Bi-Qing; Cai, Yu-Dong; Feng, Kai-Yan; Li, Zhan-Dong; Jiang, Yang

    2013-01-27

    Identification of catalytic residues plays a key role in understanding how enzymes work. Although numerous computational methods have been developed to predict catalytic residues and active sites, the prediction accuracy remains relatively low with high false positives. In this work, we developed a novel predictor based on the Random Forest algorithm (RF) aided by the maximum relevance minimum redundancy (mRMR) method and incremental feature selection (IFS). We incorporated features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure and solvent accessibility to predict active sites of enzymes and achieved an overall accuracy of 0.885687 and MCC of 0.689226 on an independent test dataset. Feature analysis showed that every category of the features except disorder contributed to the identification of active sites. It was also shown via the site-specific feature analysis that the features derived from the active site itself contributed most to the active site determination. Our prediction method may become a useful tool for identifying the active sites and the key features identified by the paper may provide valuable insights into the mechanism of catalysis.

  11. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

    The activation of molecular O 2as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O 2activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O 2dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O 2dissociationmore » is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O 2dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction.« less

  12. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

    2016-01-01

    The activation of molecular O 2as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O 2activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O 2dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O 2dissociationmore » is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O 2dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction.« less

  13. Brownian aggregation rate of colloid particles with several active sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V., E-mail: chern@ns.kinetics.nsc.ru

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shownmore » to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.« less

  14. Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

    PubMed

    Plaga, W; Frank, R; Knappe, J

    1988-12-15

    Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

  15. Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

    PubMed

    Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

    2017-05-19

    The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

  16. Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

    PubMed

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

  17. Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase.

    PubMed

    Ahn, Young O; Mahinthichaichan, Paween; Lee, Hyun Ju; Ouyang, Hanlin; Kaluka, Daniel; Yeh, Syun-Ru; Arjona, Davinia; Rousseau, Denis L; Tajkhorshid, Emad; Adelroth, Pia; Gennis, Robert B

    2014-10-21

    The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K(C)-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b3. The data suggest a linkage between residues lining the K(C)-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241(Vc) and the active site cross-linked Y255(Vc), as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

  18. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

  19. Comparative analysis of amino acid composition in the active site of nirk gene encoding copper-containing nitrite reductase (CuNiR) in bacterial spp.

    PubMed

    Adhikari, Utpal Kumar; Rahman, M Mizanur

    2017-04-01

    The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory. Copyright © 2016. Published by Elsevier Ltd.

  20. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

  1. Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

    PubMed Central

    O'Rourke, Patrick E. F.; Eadsforth, Thomas C.; Fyfe, Paul K.; Shepherd, Sharon M.; Hunter, William N.

    2011-01-01

    4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism

  2. Environmental significance of Upper Miocene phosphorites at hominid sites in the Lukeino Formation (Tugen Hills, Kenya)

    NASA Astrophysics Data System (ADS)

    Dericquebourg, Perrine; Person, Alain; Ségalen, Loïc; Pickford, Martin; Senut, Brigitte; Fagel, Nathalie

    2015-08-01

    The Lukeino Formation contains an important sedimentary and fossiliferous record of the late Miocene (6.09-5.68 Ma), which has in particular yielded the fossil remains of the oldest East African bipedal hominid called Orrorin tugenensis. This fluvio-lacustrine sedimentary succession crops out in the Kenyan part of the East African Rift. It is mainly composed of clay to sandy clay deposits intercalated with volcanic ash horizons, and localized layers of carbonates and diatomites. A detailed sedimentological and mineralogical study of the Lukeino Formation was conducted to throw light on the environmental conditions in which the hominids lived. Several centimetric, relatively continuous and indurated phosphatic horizons, of sedimentary origin, were identified at two sites (Sunbarua and Kapcheberek). Mineralogical (XRD) and geochemical analyses as well as observations by SEM, which was coupled with an energy dispersive spectroscopy (EDS) microprobe, indicate that the autochthonous phosphate layers are composed of a micritic matrix of francolite (38-93%), with incorporation of silicates in variable proportions from one layer to another. The phosphate matrix contains very well preserved and abundant diatom frustules in the basal phosphate layer. These diatoms are identified as Aulacoseira granulata, implying a pH of 7.8-8.2 for freshwaters of the Palaeolake Lukeino. Calcitic tubular structures, linked to a possible bacterial origin, are also observed locally. Phosphate layers occur abruptly within a thick clay-sandy series, associated with an intense runoff phase during the deposition of this interval of the Lukeino Formation. The massive and cyclic input of phosphorus to the lake promoted productivity to the stage where it caused a diatom bloom. The establishment of several phosphate horizons testifies to successive phases of eutrophication of Palaeolake Lukeino. The diatom cells provided some of the organic matter, which was decomposed by bacterial activity at the

  3. Motion Predicts Clinical Callus Formation

    PubMed Central

    Elkins, Jacob; Marsh, J. Lawrence; Lujan, Trevor; Peindl, Richard; Kellam, James; Anderson, Donald D.; Lack, William

    2016-01-01

    Background: Mechanotransduction is theorized to influence fracture-healing, but optimal fracture-site motion is poorly defined. We hypothesized that three-dimensional (3-D) fracture-site motion as estimated by finite element (FE) analysis would influence callus formation for a clinical series of supracondylar femoral fractures treated with locking-plate fixation. Methods: Construct-specific FE modeling simulated 3-D fracture-site motion for sixty-six supracondylar femoral fractures (OTA/AO classification of 33A or 33C) treated at a single institution. Construct stiffness and directional motion through the fracture were investigated to assess the validity of construct stiffness as a surrogate measure of 3-D motion at the fracture site. Callus formation was assessed radiographically for all patients at six, twelve, and twenty-four weeks postoperatively. Univariate and multivariate linear regression analyses examined the effects of longitudinal motion, shear (transverse motion), open fracture, smoking, and diabetes on callus formation. Construct types were compared to determine whether their 3-D motion profile was associated with callus formation. Results: Shear disproportionately increased relative to longitudinal motion with increasing bridge span, which was not predicted by our assessment of construct stiffness alone. Callus formation was not associated with open fracture, smoking, or diabetes at six, twelve, or twenty-four weeks. However, callus formation was associated with 3-D fracture-site motion at twelve and twenty-four weeks. Longitudinal motion promoted callus formation at twelve and twenty-four weeks (p = 0.017 for both). Shear inhibited callus formation at twelve and twenty-four weeks (p = 0.017 and p = 0.022, respectively). Titanium constructs with a short bridge span demonstrated greater longitudinal motion with less shear than did the other constructs, and this was associated with greater callus formation (p < 0.001). Conclusions: In this study of

  4. Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: Construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Street, I.P.; Poulter, C.D.

    1990-08-14

    Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinantmore » IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with (4-{sup 3}H)FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.« less

  5. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    PubMed Central

    Miao, Yinglong; Baudry, Jerome

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site. PMID:21943431

  6. Active Site Mutations Change the Cleavage Specificity of Neprilysin

    PubMed Central

    Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

    2012-01-01

    Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

  7. Exploring Formative Assessment Using Cultural Historical Activity Theory

    ERIC Educational Resources Information Center

    Asghar, Mandy

    2013-01-01

    Formative assessment is a pedagogic practice that has been the subject of much research and debate, as to how it can be used most effectively to deliver enhanced student learning in the higher education setting. Often described as a complex concept it embraces activities that range from facilitating students understanding of assessment standards,…

  8. THE FORMATION OF AN INVERSE S-SHAPED ACTIVE-REGION FILAMENT DRIVEN BY SUNSPOT MOTION AND MAGNETIC RECONNECTION

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yan, X. L.; Xue, Z. K.; Wang, J. C.

    2016-11-20

    We present a detailed study of the formation of an inverse S-shaped filament prior to its eruption in active region NOAA 11884 from 2013 October 31 to November 2. In the initial stage, clockwise rotation of a small positive sunspot around the main negative trailing sunspot formed a curved filament. Then the small sunspot cancelled with the negative magnetic flux to create a longer active-region filament with an inverse S-shape. At the cancellation site a brightening was observed in UV and EUV images and bright material was transferred to the filament. Later the filament erupted after cancellation of two oppositemore » polarities below the upper part of the filament. Nonlinear force-free field extrapolation of vector photospheric fields suggests that the filament may have a twisted structure, but this cannot be confirmed from the current observations.« less

  9. Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

    PubMed

    Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

    2014-06-01

    Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

  10. Active Galactic Nuclei, Host Star Formation, and the Far Infrared

    NASA Astrophysics Data System (ADS)

    Draper, Aden R.; Ballantyne, D. R.

    2011-05-01

    Telescopes like Herschel and the Atacama Large Millimeter/submillimeter Array (ALMA) are creating new opportunities to study sources in the far infrared (FIR), a wavelength region dominated by cold dust emission. Probing cold dust in active galaxies allows for study of the star formation history of active galactic nuclei (AGN) hosts. The FIR is also an important spectral region for observing AGN which are heavily enshrouded by dust, such as Compton thick (CT) AGN. By using information from deep X-ray surveys and cosmic X-ray background synthesis models, we compute Cloudy photoionization simulations which are used to predict the spectral energy distribution (SED) of AGN in the FIR. Expected differential number counts of AGN and their host galaxies are calculated in the Herschel bands. The expected contribution of AGN and their hosts to the cosmic infrared background (CIRB) is also computed. Multiple star formation scenarios are investigated using a modified blackbody star formation SED. It is found that FIR observations at 350 and 500 um are an excellent tool in determining the star formation history of AGN hosts. Additionally, the AGN contribution to the CIRB can be used to determine whether star formation in AGN hosts evolves differently than in normal galaxies. AGN and host differential number counts are dominated by CT AGN in the Herschel-SPIRE bands. Therefore, X-ray stacking of bright SPIRE sources is likely to disclose a large fraction of the CT AGN population.

  11. Alcohol Dehydrogenase Activities of Wine Yeasts in Relation to Higher Alcohol Formation

    PubMed Central

    Singh, Rajendra; Kunkee, Ralph E.

    1976-01-01

    Alcohol dehydrogenase activities were examined in cell-free extracts of 10 representative wine yeast strains having various productivities of higher alcohols (fusel oil). The amount of fusel alcohols (n-propanol, isobutanol, active pentanol, and isopentanol) produced by the different yeasts and the specific alcohol dehydrogenase activities with the corresponding alcohols as substrates were found to be significantly related. No such relationship was found for ethanol. The amounts of higher alcohols formed during vinification could be predicted from the specific activities of the alcohol dehydrogenases with high accuracy. The results suggest a close relationship between the control of the activities of alcohol dehydrogenase and the formation of fusel oil alcohols. Also, new procedures for the prediction of higher alcohol formation during alcoholic beverage fermentation are suggested. PMID:16345179

  12. BMP-2 and titanium particles synergistically activate osteoclast formation

    PubMed Central

    Sun, S.X.; Guo, H.H.; Zhang, J.; Yu, B.; Sun, K.N.; Jin, Q.H.

    2014-01-01

    A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation. PMID:24820069

  13. Can we define an asymptotic value for the ice active surface site density for heterogeneous ice nucleation?

    NASA Astrophysics Data System (ADS)

    Niedermeier, Dennis; Augustin-Bauditz, Stefanie; Hartmann, Susan; Wex, Heike; Ignatius, Karoliina; Stratmann, Frank

    2015-04-01

    The formation of ice in atmospheric clouds has a substantial influence on the radiative properties of clouds as well as on the formation of precipitation. Therefore much effort has been made to understand and quantify the major ice formation processes in clouds. Immersion freezing has been suggested to be a dominant primary ice formation process in low and mid-level clouds (mixed-phase cloud conditions). It also has been shown that mineral dust particles are the most abundant ice nucleating particles in the atmosphere and thus may play an important role for atmospheric ice nucleation (Murray et al., 2012). Additionally, biological particles like bacteria and pollen are suggested to be potentially involved in atmospheric ice formation, at least on a regional scale (Murray et al., 2012). In recent studies for biological particles (SNOMAX and birch pollen), it has been demonstrated that freezing is induced by ice nucleating macromolecules and that an asymptotic value for the mass density of these ice nucleating macromolecules can be determined (Hartmann et al., 2013; Augustin et al., 2013, Wex et al., 2014). The question arises whether such an asymptotic value can also be determined for the ice active surface site density ns, a parameter which is commonly used to describe the ice nucleation activity of e.g., mineral dust. Such an asymptotic value for ns could be an important input parameter for atmospheric modeling applications. In the presented study, we therefore investigated the immersion freezing behavior of droplets containing size-segregated, monodisperse feldspar particles utilizing the Leipzig Aerosol Cloud Interaction Simulator (LACIS). For all particle sizes considered in the experiments, we observed a leveling off of the frozen droplet fraction reaching a plateau within the heterogeneous freezing temperature regime (T > -38°C) which was proportional to the particle surface area. Based on these findings, we could determine an asymptotic value for the ice

  14. Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

    PubMed

    Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

    2017-02-27

    The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

  15. Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

    PubMed

    Ali, Abid; Azam, Mohd W; Khan, Asad U

    2018-06-01

    New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Ventifact Formation in the Mojave Desert: Field Analogs for Martian Processes

    NASA Technical Reports Server (NTRS)

    Laity, J. E.; Bridges, N. T.; Boyle, T. K.

    2001-01-01

    Two field studies in the Mojave Desert, California, shed light on processes of ventifact formation. The field sites are located on a ridge at Little Cowhole Mountain, which lies approximately 12 km south of Baker, and on an unnamed ridge situated along the northern boundary of the Mojave River Sink (Rasor Road site). The rocks at Little Cowhole Mountain are a blue-grey marble/dolomite, whereas those at Rasor Road are Miocene volcanic rocks (basalt). At both sites the abrasive agent is a fine-grained aeolian sand which was probably derived largely from the Mojave River. There are minimal modem inputs of sand to either site: abrasion occurs as a result of unique climatic and topographic conditions which allow pre-existing sand to be recycled from one aspect of the ridge to the other. Climatic conditions are well suited for ventifact formation. Owing, to the dry climate (marked by low average relative humidity, infrequent dew, and low annual rainfall), rates of chemical weathering are low. Where resurfacing of the rocks by sand abrasion proceeds at a rate greater than weathering, the ventifacts are considered "active." Active ventifacts are found atop and straddling the ridge crests, in the zone of maximum wind velocity and sediment supply. Inactive ventifacts occur where modem weathering Processes exceed abrasion rates; principally on the basal two-thirds of the hillslope, where wind velocity and sediment supply are lower. At intermediate locations between the slope base and crest, ventifacts are either active or inactive, depending on local conditions. The presence of relict ventifacts at the study sites, as well as elsewhere in the eastern Mojave Desert, suggests that the conditions for venti fact formation must have been more intense and extensive in the past. Together, the studies illustrate that the processes that interact to form ventifacts are highly complex, and must be studied at many scales. Small-scale effects, such as local topography, plant cover, or

  17. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

    PubMed

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2010-10-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

  19. Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis.

    PubMed

    MacGrogan, Donal; D'Amato, Gaetano; Travisano, Stanislao; Martinez-Poveda, Beatriz; Luxán, Guillermo; Del Monte-Nieto, Gonzalo; Papoutsi, Tania; Sbroggio, Mauro; Bou, Vanesa; Gomez-Del Arco, Pablo; Gómez, Manuel Jose; Zhou, Bin; Redondo, Juan Miguel; Jiménez-Borreguero, Luis J; de la Pompa, José Luis

    2016-05-13

    The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. The aim of this study is to determine the functional specificity of Notch in valve development. Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart

  20. Active Iron Sites of Disordered Mesoporous Silica Catalyst FeKIL-2 in the Oxidation of Volatile Organic Compounds (VOC)

    PubMed Central

    Rangus, Mojca; Mazaj, Matjaž; Dražić, Goran; Popova, Margarita; Tušar, Nataša Novak

    2014-01-01

    Iron-functionalized disordered mesoporous silica (FeKIL-2) is a promising, environmentally friendly, cost-effective and highly efficient catalyst for the elimination of volatile organic compounds (VOCs) from polluted air via catalytic oxidation. In this study, we investigated the type of catalytically active iron sites for different iron concentrations in FeKIL-2 catalysts using advanced characterization of the local environment of iron atoms by a combination of X-ray Absorption Spectroscopy Techniques (XANES, EXAFS) and Atomic-Resolution Scanning Transmission Electron Microscopy (AR STEM). We found that the molar ratio Fe/Si ≤ 0.01 leads to the formation of stable, mostly isolated Fe3+ sites in the silica matrix, while higher iron content Fe/Si > 0.01 leads to the formation of oligonuclear iron clusters. STEM imaging and EELS techniques confirmed the existence of these clusters. Their size ranges from one to a few nanometers, and they are unevenly distributed throughout the material. The size of the clusters was also found to be similar, regardless of the nominal concentration of iron (Fe/Si = 0.02 and Fe/Si = 0.05). From the results obtained from sample characterization and model catalytic tests, we established that the enhanced activity of FeKIL-2 with the optimal Fe/Si = 0.01 ratio can be attributed to: (1) the optimal concentration of stable isolated Fe3+ in the silica support; and (2) accelerated diffusion of the reactants in disordered mesoporous silica (FeKIL-2) when compared to ordered mesoporous silica materials (FeSBA-15, FeMCM-41). PMID:28788674

  1. Patient-reported physical activity questionnaires: A systematic review of content and format

    PubMed Central

    2012-01-01

    Background Many patients with chronic illness are limited in their physical activities. This systematic review evaluates the content and format of patient-reported outcome (PRO) questionnaires that measure physical activity in elderly and chronically ill populations. Methods Questionnaires were identified by a systematic literature search of electronic databases (Medline, Embase, PsychINFO & CINAHL), hand searches (reference sections and PROQOLID database) and expert input. A qualitative analysis was conducted to assess the content and format of the questionnaires and a Venn diagram was produced to illustrate this. Each stage of the review process was conducted by at least two independent reviewers. Results 104 questionnaires fulfilled our criteria. From these, 182 physical activity domains and 1965 items were extracted. Initial qualitative analysis of the domains found 11 categories. Further synthesis of the domains found 4 broad categories: 'physical activity related to general activities and mobility', 'physical activity related to activities of daily living', 'physical activity related to work, social or leisure time activities', and '(disease-specific) symptoms related to physical activity'. The Venn diagram showed that no questionnaires covered all 4 categories and that the '(disease-specific) symptoms related to physical activity' category was often not combined with the other categories. Conclusions A large number of questionnaires with a broad range of physical activity content were identified. Although the content could be broadly organised, there was no consensus on the content and format of physical activity PRO questionnaires in elderly and chronically ill populations. Nevertheless, this systematic review will help investigators to select a physical activity PRO questionnaire that best serves their research question and context. PMID:22414164

  2. Regulation of invadopodia formation and activity by CD147

    PubMed Central

    Grass, G. Daniel; Bratoeva, Momka; Toole, Bryan P.

    2012-01-01

    A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia to breach these structural barriers. Invadopodia are actin-based, lipid-raft-enriched membrane protrusions containing membrane-type-1 matrix metalloproteinase (MT1-MMP; also known as matrix metalloproteinase 14; MMP14) and several signaling proteins. CD147 (emmprin, basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in many systems. In this study we show that upregulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodia-like structures and co-fractionate in membrane compartments with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, probably through assembly of MT1-MMP-containing complexes within lipid-raft domains of the invadopodia. PMID:22389410

  3. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

    PubMed

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-12-16

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

  4. Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

    PubMed

    Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

    2008-07-16

    Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

  5. Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

    PubMed Central

    Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

    2008-01-01

    Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS. PMID:18629001

  6. Effect of phosphate activating group on oligonucleotide formation on montmorillonite: the regioselective formation of 3',5'-linked oligoadenylates

    NASA Technical Reports Server (NTRS)

    Prabahar, K. J.; Cole, T. D.; Ferris, J. P.

    1994-01-01

    The effects of amine structure on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidates of adenosine are investigated. 4-Aminopyridine derivatives yielded oligoadenylates as long as dodecamers with a regioselectivity for 3',5'-phosphodiester bond formation averaging 88%. Linear and cyclic oligomers are obtained and no A5'ppA-containing products are detected. Oligomers as long as the hexanucleotide are obtained using 2-aminobenzimidazole as the activating group. A predominance of pA2'pA is detected in the dimer fraction along with cyclic 3',5'-trimer; no A5'ppA-containing oligomers were detected. Little or no oligomer formation was observed when morpholine, piperidine, pyrazole, 1,2,4-triazole, and 2-pyridone are used as phosphate-activating groups. The effects of the structure of the phosphate activating group on the oligomer structure and chain lengths are discussed.

  7. Incorporation of RANKL promotes osteoclast formation and osteoclast activity on β-TCP ceramics.

    PubMed

    Choy, John; Albers, Christoph E; Siebenrock, Klaus A; Dolder, Silvia; Hofstetter, Wilhelm; Klenke, Frank M

    2014-12-01

    β-Tricalcium phosphate (β-TCP) ceramics are approved for the repair of osseous defects. In large defects, however, the substitution of the material by authentic bone is inadequate to provide sufficient long-term mechanical stability. We aimed to develop composites of β-TCP ceramics and receptor activator of nuclear factor κ-B ligand (RANKL) to enhance the formation of osteoclasts and promote cell mediated calcium phosphate resorption. RANKL was adsorbed superficially onto β-TCP ceramics or incorporated into a crystalline layer of calcium phosphate by the use of a co-precipitation technique. Murine osteoclast precursors were seeded onto the ceramics. After 15 days, the formation of osteoclasts was quantified cytologically and colorimetrically with tartrate-resistant acidic phosphatase (TRAP) staining and TRAP activity measurements, respectively. Additionally, the expression of transcripts encoding the osteoclast gene products cathepsin K, calcitonin receptor, and of the sodium/hydrogen exchanger NHA2 were quantified by real-time PCR. The activity of newly formed osteoclasts was evaluated by means of a calcium phosphate resorption assay. Superficially adsorbed RANKL did not induce the formation of osteoclasts on β-TCP ceramics. When co-precipitated onto β-TCP ceramics RANKL supported the formation of mature osteoclasts. The development of osteoclast lineage cells was further confirmed by the increased expression of cathepsin K, calcitonin receptor, and NHA2. Incorporated RANKL stimulated the cells to resorb crystalline calcium phosphate. Our in vitro study shows that RANKL incorporated into β-TCP ceramics induces the formation of active, resorbing osteoclasts on the material surface. Once formed, osteoclasts mediate the release of RANKL thereby perpetuating their differentiation and activation. In vivo, the stimulation of osteoclast-mediated resorption may contribute to a coordinated sequence of material resorption and bone formation. Further in vivo studies

  8. Photoproduct formation of endogeneous protoporphyrin and its photodynamic activity

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Schneckenburger, Herbert; Rueck, Angelika C.; Auchter, S.

    1991-11-01

    Human skin shows a strong autofluorescence in the red spectral region caused on the porphyrin production of the Gram positive lipophile skin bacterium Propionibacterium acnes. Irradiation of these bacteria reduces the integral fluorescence intensity and induces the formation of fluorescent photoproducts. The fluorescence band at around 670 nm and the decay times of around 1 ns and 5 ns are typical for protoporphyrin products. The photoproduct formation is connected with an increased absorption in the red spectral region. However the photodynamic activity of these photoproducts determined by scattering measurements on human erythrocytes is lower than that of protoporphyrin IX. 1:

  9. Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

    PubMed Central

    Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

    1992-01-01

    By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

  10. Sediment infilling and wetland formation dynamics in an active crevasse splay of the Mississippi River delta

    USGS Publications Warehouse

    Cahoon, Donald R.; White, David A.; Lynch, James C.

    2011-01-01

    Crevasse splay environments provide a mesocosm for evaluating wetland formation and maintenance processes on a decadal time scale. Site elevation, water levels, vertical accretion, elevation change, shallow subsidence, and plant biomass were measured at five habitats along an elevation gradient to evaluate wetland formation and development in Brant Pass Splay; an active crevasse splay of the Balize delta of the Mississippi River. The processes of vertical development (vertical accretion, elevation change, and shallow subsidence) were measured with the surface elevation table–marker horizon method. There were three distinct stages to the accrual of elevation capital and wetland formation in the splay: sediment infilling, vegetative colonization, and development of a mature wetland community. Accretion, elevation gain, and shallow subsidence all decreased by an order of magnitude from the open water (lowest elevation) to the forest (highest elevation) habitats. Vegetative colonization occurred within the first growing season following emergence of the mud surface. An explosively high rate of below-ground production quickly stabilized the loosely consolidated sub-aerial sediments. After emergent vegetation colonization, vertical development slowed and maintenance of marsh elevation was driven both by sediment trapping by the vegetation and accumulation of plant organic matter in the soil. Continued vertical development and survival of the marsh then depended on the health and productivity of the plant community. The process of delta wetland formation is both complex and nonlinear. Determining the dynamics of wetland formation will help in understanding the processes driving the past building of the delta and in developing models for restoring degraded wetlands in the Mississippi River delta and other deltas around the world.

  11. P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation.

    PubMed

    Théorêt, Jean-François; Yacoub, Daniel; Hachem, Ahmed; Gillis, Marc-Antoine; Merhi, Yahye

    2011-09-01

    Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology. Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P(-/-)) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α(IIb)β(3). Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P(-/-) mice. In addition, CD62P(-/-) mice exhibited thrombosis abnormalities without an α(IIb)β(3) activation defect. This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Formation of p-type ZnO thin film through co-implantation

    NASA Astrophysics Data System (ADS)

    Chuang, Yao-Teng; Liou, Jhe-Wei; Woon, Wei-Yen

    2017-01-01

    We present a study on the formation of p-type ZnO thin film through ion implantation. Group V dopants (N, P) with different ionic radii are implanted into chemical vapor deposition grown ZnO thin film on GaN/sapphire substrates prior to thermal activation. It is found that mono-doped ZnO by N+ implantation results in n-type conductivity under thermal activation. Dual-doped ZnO film with a N:P ion implantation dose ratio of 4:1 is found to be p-type under certain thermal activation conditions. Higher p-type activation levels (1019 cm-3) under a wider thermal activation range are found for the N/P dual-doped ZnO film co-implanted by additional oxygen ions. From high resolution x-ray diffraction and x-ray photoelectron spectroscopy it is concluded that the observed p-type conductivities are a result of the promoted formation of PZn-4NO complex defects via the concurrent substitution of nitrogen at oxygen sites and phosphorus at zinc sites. The enhanced solubility and stability of acceptor defects in oxygen co-implanted dual-doped ZnO film are related to the reduction of oxygen vacancy defects at the surface. Our study demonstrates the prospect of the formation of stable p-type ZnO film through co-implantation.

  13. Efficient CO2-Reducing Activity of NAD-Dependent Formate Dehydrogenase from Thiobacillus sp. KNK65MA for Formate Production from CO2 Gas

    PubMed Central

    Cho, Dae Haeng; Kim, Min Hoo; Lee, Sang Hyun; Jung, Kwang Deog; Kim, Yong Hwan

    2014-01-01

    NAD-dependent formate dehydrogenase (FDH) from Candida boidinii (CbFDH) has been widely used in various CO2-reduction systems but its practical applications are often impeded due to low CO2-reducing activity. In this study, we demonstrated superior CO2-reducing properties of FDH from Thiobacillus sp. KNK65MA (TsFDH) for production of formate from CO2 gas. To discover more efficient CO2-reducing FDHs than a reference enzyme, i.e. CbFDH, five FDHs were selected with biochemical properties and then, their CO2-reducing activities were evaluated. All FDHs including CbFDH showed better CO2-reducing activities at acidic pHs than at neutral pHs and four FDHs were more active than CbFDH in the CO2 reduction reaction. In particular, the FDH from Thiobacillus sp. KNK65MA (TsFDH) exhibited the highest CO2-reducing activity and had a dramatic preference for the reduction reaction, i.e., a 84.2-fold higher ratio of CO2 reduction to formate oxidation in catalytic efficiency (k cat/K B) compared to CbFDH. Formate was produced from CO2 gas using TsFDH and CbFDH, and TsFDH showed a 5.8-fold higher formate production rate than CbFDH. A sequence and structural comparison showed that FDHs with relatively high CO2-reducing activities had elongated N- and C-terminal loops. The experimental results demonstrate that TsFDH can be an alternative to CbFDH as a biocatalyst in CO2 reduction systems. PMID:25061666

  14. Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

    PubMed

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-04-20

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

  15. Fgf signaling controls pharyngeal taste bud formation through miR-200 and Delta-Notch activity.

    PubMed

    Kapsimali, Marika; Kaushik, Anna-Lila; Gibon, Guillaume; Dirian, Lara; Ernest, Sylvain; Rosa, Frederic M

    2011-08-01

    Taste buds, the taste sensory organs, are conserved in vertebrates and composed of distinct cell types, including taste receptor, basal/presynaptic and support cells. Here, we characterize zebrafish taste bud development and show that compromised Fgf signaling in the larva results in taste bud reduction and disorganization. We determine that Fgf activity is required within pharyngeal endoderm for formation of Calb2b(+) cells and reveal miR-200 and Delta-Notch signaling as key factors in this process. miR-200 knock down shows that miR-200 activity is required for taste bud formation and in particular for Calb2b(+) cell formation. Compromised delta activity in mib(-/-) dramatically reduces the number of Calb2b(+) cells and increases the number of 5HT(+) cells. Conversely, larvae with increased Notch activity and ascl1a(-/-) mutants are devoid of 5HT(+) cells, but have maintained and increased Calb2b(+) cells, respectively. These results show that Delta-Notch signaling is required for intact taste bud organ formation. Consistent with this, Notch activity restores Calb2b(+) cell formation in pharyngeal endoderm with compromised Fgf signaling, but fails to restore the formation of these cells after miR-200 knock down. Altogether, this study provides genetic evidence that supports a novel model where Fgf regulates Delta-Notch signaling, and subsequently miR-200 activity, in order to promote taste bud cell type differentiation.

  16. Synthesis of a molecularly defined single-active site heterogeneous catalyst for selective oxidation of N-heterocycles.

    PubMed

    Zhang, Yujing; Pang, Shaofeng; Wei, Zhihong; Jiao, Haijun; Dai, Xingchao; Wang, Hongli; Shi, Feng

    2018-04-13

    Generally, a homogeneous catalyst exhibits good activity and defined active sites but it is difficult to recycle. Meanwhile, a heterogeneous catalyst can easily be reused but its active site is difficult to reveal. It is interesting to bridge the gap between homogeneous and heterogeneous catalysis via controllable construction of a heterogeneous catalyst containing defined active sites. Here, we report that a molecularly defined, single-active site heterogeneous catalyst has been designed and prepared via the oxidative polymerization of maleimide derivatives. These polymaleimide derivatives can be active catalysts for the selective oxidation of heterocyclic compounds to quinoline and indole via the recycling of -C=O and -C-OH groups, which was confirmed by tracing the reaction with GC-MS using maleimide as the catalyst and by FT-IR analysis with polymaleimide as the catalyst. These results might promote the development of heterogeneous catalysts with molecularly defined single active sites exhibiting a comparable activity to homogeneous catalysts.

  17. Psycho-Pedagogical Conditions of Formation of Professional Creative Activity of Future Professionals

    ERIC Educational Resources Information Center

    Askarovna, Uzakbayeva Sakypzhamal; Yertaevna, Abeltayeva Zhanel; Erhanovna, Sadykova Ayzhan; Rysbekova, R.

    2015-01-01

    Organizational, psychological and pedagogical conditions (the position of student in the educational process, the inclusion of students in active and independent activities, the creation of a positive creative environment and psychological climate, the active use of the forms, methods, technologies, adequate formation of professional creative…

  18. Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

    PubMed

    Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

    2018-05-09

    Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  19. Impacts of glycolate and formate radiolysis and thermolysis on hydrogen generation rate calculations for the Savannah River Site tank farm

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Crawford, C. L.; King, W. D.

    Savannah River Remediation (SRR) personnel requested that the Savannah River National Laboratory (SRNL) evaluate available data and determine its applicability to defining the impact of planned glycolate anion additions to Savannah River Site (SRS) High Level Waste (HLW) on Tank Farm flammability (primarily with regard to H 2 production). Flammability evaluations of formate anion, which is already present in SRS waste, were also needed. This report describes the impacts of glycolate and formate radiolysis and thermolysis on Hydrogen Generation Rate (HGR) calculations for the SRS Tank Farm.

  20. Interactive flare sites within an active region complex

    NASA Technical Reports Server (NTRS)

    Poletto, G.; Gary, G. A.; Machado, M. E.

    1993-01-01

    We examine here a set of images of an active region complex, acquired on June 24-25, 1980, by the Hard X-ray Imaging Spectrometer on SMM, with the purpose of establishing whether there was any interplay between the frequent activity observed at different sites in the activity center and, in such a case, how the interaction was established. By analyzing both quiet and active orbits we show that, as a rule, activity originating in one region triggers the other region's activity. However, we find little unambiguous evidence for the presence of large-scale interconnecting loops. A comparison of X-ray images with magnetic field observations suggested that we interpret the active region behavior in terms of the interaction between different loop systems, in a scenario quite analogous to the interacting bipole representation of individual flares. We conclude that active region interplay provides an easily observable case to study the time-dependent topology and the mechanisms for the spreading of activity in transient events over all energy scales.

  1. Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

    PubMed

    Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2016-04-01

    Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

  2. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    PubMed

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  3. Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure*

    PubMed Central

    Hervé, Guy; Evans, Hedeel Guy; Fernado, Roshini; Patel, Chandni; Hachem, Fatme; Evans, David R.

    2017-01-01

    Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues. PMID:27746403

  4. Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

    PubMed Central

    Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

    2015-01-01

    Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

  5. Engineering streptokinase for generation of active site-labeled plasminogen analogs*

    PubMed Central

    Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

    2011-01-01

    We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

  6. Correlation of surface site formation to nanoisland growth in the electrochemical roughening of Pt(111)

    NASA Astrophysics Data System (ADS)

    Jacobse, Leon; Huang, Yi-Fan; Koper, Marc T. M.; Rost, Marcel J.

    2018-03-01

    Platinum plays a central role in a variety of electrochemical devices and its practical use depends on the prevention of electrode degradation. However, understanding the underlying atomic processes under conditions of repeated oxidation and reduction inducing irreversible surface structure changes has proved challenging. Here, we examine the correlation between the evolution of the electrochemical signal of Pt(111) and its surface roughening by simultaneously performing cyclic voltammetry and in situ electrochemical scanning tunnelling microscopy (EC-STM). We identify a `nucleation and early growth' regime of nanoisland formation, and a `late growth' regime after island coalescence, which continues up to at least 170 cycles. The correlation analysis shows that each step site that is created in the `late growth' regime contributes equally strongly to both the electrochemical and the roughness evolution. In contrast, in the `nucleation and early growth' regime, created step sites contribute to the roughness, but not to the electrochemical signal.

  7. Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

    PubMed

    Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

    2018-06-15

    The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The multidisciplinary treatment plan: a format for enhancing activity therapy department involvement.

    PubMed

    Lang, E; Mattson, M

    1985-01-01

    A structured, goal-oriented format for enhancing the involvement of activity therapy disciplines in the multidisciplinary treatment planning process has been developed in a large private psychiatric teaching hospital. The format, an adaptation of the problem-oriented record, encompasses formal procedures for identifying and recording relevant problems, goals, methods, and objectives for activity therapy treatment. The benefits of this approach include the development of specific, measurable, attainable functional goals; increased accountability in treatment planning and delivery; less time spent in documentation; and education of other staff about the role and function of activities therapy. Patients have a better understanding of their goals and the steps needed to achieve them and show increased participation in the therapy process.

  9. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  10. Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

    PubMed Central

    tenBrink, Felix; Schink, Bernhard; Kroneck, Peter M. H.

    2011-01-01

    The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme. PMID:21193613

  11. Comparison of the active-site design of molybdenum oxo-transfer enzymes by quantum mechanical calculations.

    PubMed

    Li, Jilai; Ryde, Ulf

    2014-11-17

    There are three families of mononuclear molybdenum enzymes that catalyze oxygen atom transfer (OAT) reactions, named after a typical example from each family, viz., dimethyl sulfoxide reductase (DMSOR), sulfite oxidase (SO), and xanthine oxidase (XO). These families differ in the construction of their active sites, with two molybdopterin groups in the DMSOR family, two oxy groups in the SO family, and a sulfido group in the XO family. We have employed density functional theory calculations on cluster models of the active sites to understand the selection of molybdenum ligands in the three enzyme families. Our calculations show that the DMSOR active site has a much stronger oxidative power than the other two sites, owing to the extra molybdopterin ligand. However, the active sites do not seem to have been constructed to make the OAT reaction as exergonic as possible, but instead to keep the reaction free energy close to zero (to avoid excessive loss of energy), thereby making the reoxidation (SO and XO) or rereduction of the active sites (DMSOR) after the OAT reaction facile. We also show that active-site models of the three enzyme families can all catalyze the reduction of DMSO and that the DMSOR model does not give the lowest activation barrier. Likewise, all three models can catalyze the oxidation of sulfite, provided that the Coulombic repulsion between the substrate and the enzyme model can be overcome, but for this harder reaction, the SO model gives the lowest activation barrier, although the differences are not large. However, only the XO model can catalyze the oxidation of xanthine, owing to its sulfido ligand.

  12. Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

    PubMed

    Bate, Paul; Warwicker, Jim

    2004-07-02

    Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

  13. Cells in the monkey ponto-medullary reticular formation modulate their activity with slow finger movements

    PubMed Central

    Soteropoulos, Demetris S; Williams, Elizabeth R; Baker, Stuart N

    2012-01-01

    Recent work has shown that the primate reticulospinal tract can influence spinal interneurons and motoneurons involved in control of the hand. However, demonstrating connectivity does not reveal whether reticular outputs are modulated during the control of different types of hand movement. Here, we investigated how single unit discharge in the pontomedullary reticular formation (PMRF) modulated during performance of a slow finger movement task in macaque monkeys. Two animals performed an index finger flexion–extension task to track a target presented on a computer screen; single units were recorded both from ipsilateral PMRF (115 cells) and contralateral primary motor cortex (M1, 210 cells). Cells in both areas modulated their activity with the task (M1: 87%, PMRF: 86%). Some cells (18/115 in PMRF; 96/210 in M1) received sensory input from the hand, showing a short-latency modulation in their discharge following a rapid passive extension movement of the index finger. Effects in ipsilateral electromyogram to trains of stimuli were recorded at 45 sites in the PMRF. These responses involved muscles controlling the digits in 13/45 sites (including intrinsic hand muscles, 5/45 sites). We conclude that PMRF may contribute to the control of fine finger movements, in addition to its established role in control of more proximal limb and trunk movements. This finding may be especially important in understanding functional recovery after brain lesions such as stroke. PMID:22641776

  14. Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

    PubMed

    Fox, B G; Liu, Y; Dege, J E; Lipscomb, J D

    1991-01-05

    Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the

  15. Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form.

    PubMed

    Lee, Yie-Vern; Choi, Sy Bing; Wahab, Habibah A; Choong, Yee Siew

    2017-09-25

    Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C 2 -C 3 bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.

  16. Opinion formation in a social network: The role of human activity

    NASA Astrophysics Data System (ADS)

    Grabowski, Andrzej

    2009-03-01

    The model of opinion formation in human population based on social impact theory is investigated numerically. On the basis of a database received from the on-line game server, we examine the structure of social network and human dynamics. We calculate the activity of individuals, i.e. the relative time devoted daily to interactions with others in the artificial society. We study the influence of correlation between the activity of an individual and its connectivity on the process of opinion formation. We find that such correlations have a significant influence on the temperature of the phase transition and the effect of the mass media, modeled as an external stimulation acting on the social network.

  17. Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

    PubMed Central

    Hatanaka, M; Del Giudice, R; Long, C

    1975-01-01

    Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

  18. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

  19. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiO x domains are titrated on TiO x–SiO 2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiO x, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H 2O 2 activation occurs. We use this method to determine the active sitemore » densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h -1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.« less

  20. Modulation of Platelet Activation and Thrombus Formation Using a Pan-PI3K Inhibitor S14161

    PubMed Central

    Ren, Lijie; Liu, Xiaohui; Wang, Qi; He, Sudan; Wu, Qingyu; Hu, Hu; Mao, Xinliang; Zhu, Li

    2014-01-01

    The phosphatidylinositol 3–kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. Flow cytometric studies showed that S14161 inhibited convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3β at Ser9 in response to collagen, thrombin, or U46619. Comparable studies showed that S14161 has a higher potential bioavailability than LY294002, a prototypical inhibitor of pan-class I PI3K. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.05±0.99 min, n = 9) compared to the vehicle controls (3.72±0.95 min, n = 8) (P<0.05), but did not prolong the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation without significant bleeding tendency and toxicity, and considering its potential higher bioavailability, it may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. PMID:25115838

  1. Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

    PubMed Central

    Wood, Jeremy P.; Silveira, Jay R.; Maille, Nicole M.; Haynes, Laura M.

    2011-01-01

    Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. PMID:21131592

  2. Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

    PubMed

    Wood, Jeremy P; Silveira, Jay R; Maille, Nicole M; Haynes, Laura M; Tracy, Paula B

    2011-02-03

    Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

  3. Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

    Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

  4. Identification and characterization of the sodium-binding site of activated protein C.

    PubMed

    He, X; Rezaie, A R

    1999-02-19

    Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

  5. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    PubMed Central

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  6. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  7. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  8. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  9. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

    DOE PAGES

    Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

    2017-07-07

    Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

  10. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tomkinson, B.; Wernstedt, C.; Hellman, U.

    1987-11-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residuesmore » 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.« less

  11. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

    PubMed Central

    Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

  12. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

  13. Quarries of Culture: An Ethnohistorical and Environmental Account of Sacred Sites and Rock Formations in Southern California's Mission Indian Country

    ERIC Educational Resources Information Center

    Karr, Steven M.

    2005-01-01

    Sacred sites and Rock Formations throughout Southern California's India Country are described by Indians as ancestral markers, origin and place-name locales, areas of deity habitation, and power sources. Early ethnographers were keen to record the traditional stories and meanings related to them by their Native collaborators. Rock formations…

  14. A description of the index of active Florida water data collection stations and a user's guide for station or site information retrieval using computer program Findex H578

    USGS Publications Warehouse

    Merritt, M.L.

    1977-01-01

    A computerized index of water-data collection activities and retrieval software to generate publication list of this information was developed for Florida. This system serves a vital need in the administration of the many and diverse water-data collection activities. Previously, needed data was very difficult to assemble for use in program planning or project implementation. Largely descriptive, the report tells how a file of computer card images has been established which contains entries for all sites in Florida at which there is currently a water-data-collection activity. Entries include information such as identification number, station name, location, type of site, county, information about data collection, funding, and other pertinent details. The computer program FINDEX selectively retrieves entries and lists them in a format suitable for publication. Updating the index is done routinely. (Woodard-USGS)

  15. Geology, coal resources, and chemical analyses of coal from the Fruitland Formation, Kimbeto EMRIA study site, San Juan County, New Mexico

    USGS Publications Warehouse

    Schneider, Gary B.; Hildebrand, Rick T.; Affolter, Ronald H.

    1979-01-01

    The Kimbeto EMRIA study site, an area of about 20 square miles (52 km2), is located on the south margin of the San Juan Basin on the gently northward-dipping strata of the Upper Cretaceous Fruitland Formation and the Kirtland Shale. The coal beds are mainly in the lower 150 feet (45 m) of the Fruitland Format ion. Coal resources--measured, indicated, and inferred--with less than 400 feet (120 m) of overburden in the site are 69,085,000 short tons (62,660,100 metric tons), 369,078,000 short tons (334,754,000 metric tons), and 177,803,000 short tons (161,267,000 metric tons) respectively. About 68 percent of these resources are overlain by 200 feet (60 m) or less of overburden. The apparent rank of the coal ranges from subbituminous B to subbituminous A. The average Btu/lb value of 14 core samples from the site on the as-received basis is 8,240 (4580 Kcal/kg), average ash content is 23.4 percent, and average sulfur content is 0.5 percent. Analyses of coal from the Kimbeto EMRIA study site show significantly higher ash content and significantly lower contents of volatile matter, fixed carbon, carbon, and a significantly lower heat of combustion when compared with other coal analyses from the Rocky Mountain province.

  16. Active site proton delivery and the lyase activity of human CYP17A1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbonmore » scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which

  17. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  18. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    PubMed

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

  20. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  1. Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

    PubMed

    Sen, Kakali; Horrell, Sam; Kekilli, Demet; Yong, Chin W; Keal, Thomas W; Atakisi, Hakan; Moreau, David W; Thorne, Robert E; Hough, Michael A; Strange, Richard W

    2017-07-01

    Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

  2. Seasonal development of cambial activity in relation to xylem formation in Chinese fir.

    PubMed

    Wu, Hongyang; Xu, Huimin; Li, Hanyin; Wei, Dongmei; Lin, Jinxing; Li, Xiaojuan

    2016-05-20

    The vascular cambium is a lateral meristem which can differentiate into secondary phloem and xylem. The secondary growth of woody plants resulting from vascular cambium activity has been a focus of considerable attention, but the quantitative relationships between cambial activity and secondary xylem formation have been little studied. Our analysis of cytological changes in the cambium of Chinese fir (Cunninghamia lanceolata), revealed a significant positive correlation between vascular cambium cell numbers and cambium zone width through the seasonal cycle. Cambium cell numbers and the cambium cell radial diameter were closely related to xylem formation. Immuno-labeling showed that de-esterified homogalacturonan and (1-4)-β-d-galactan epitopes were highly abundant in cell walls of dormant-stage cambium, whereas high methylesterified homogalacturonan was strongly labeled in the active stage. Raman spectroscopy detected significant changes in the chemical composition of cell walls during the active-dormant stage transition. More pectin and less monolignols occurred in radial cell walls than in tangential walls during the dormant stage, but no significant changes were found in other stages, indicating that pectin accumulation facilitates cell wall expansion, with cambium activity transition. Our quantitative analysis of the relationship between cambial activity and xylem formation, as well as the cell wall modification during the active stage provides useful information about cambial characteristics and xylogenesis. Copyright © 2016. Published by Elsevier GmbH.

  3. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    DOE PAGES

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open'more » structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. In conclusion, as a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

  4. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

    PubMed

    Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  5. A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

    PubMed

    Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

    2018-06-01

    The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

    PubMed

    Fenwick, Michael K; Mehta, Angad P; Zhang, Yang; Abdelwahed, Sameh H; Begley, Tadhg P; Ealick, Steven E

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  7. [Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

    PubMed

    Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

    2017-08-25

    Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

  8. Endogenously Generated Plasmin at the Vascular Wall Injury Site Amplifies Lysine Binding Site-Dependent Plasminogen Accumulation in Microthrombi

    PubMed Central

    Brzoska, Tomasz; Tanaka-Murakami, Aki; Suzuki, Yuko; Sano, Hideto; Kanayama, Naohiro; Urano, Tetsumei

    2015-01-01

    The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation. PMID:25806939

  9. A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

    PubMed

    Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

    2016-10-04

    Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

  10. Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

    PubMed

    Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

    2013-08-21

    The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

  11. The Effect of Surface Site Ensembles on the Activity and Selectivity of Ethanol Electrooxidation by Octahedral PtNiRh Nanoparticles.

    PubMed

    Erini, Nina; Beermann, Vera; Gocyla, Martin; Gliech, Manuel; Heggen, Marc; Dunin-Borkowski, Rafal E; Strasser, Peter

    2017-06-01

    Direct ethanol fuel cells are attractive power sources based on a biorenewable, high energy-density fuel. Their efficiency is limited by the lack of active anode materials which catalyze the breaking of the C-C bond coupled to the 12-electron oxidation to CO 2 . We report shape-controlled PtNiRh octahedral ethanol oxidation electrocatalysts with excellent activity and previously unachieved low onset potentials as low as 0.1 V vs. RHE, while being highly selective to complete oxidation to CO 2 . Our comprehensive characterization and in situ electrochemical ATR studies suggest that the formation of a ternary surface site ensemble around the octahedral Pt 3 Ni 1 Rh x nanoparticles plays a crucial mechanistic role for this behavior. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Well-Defined Metal-O6 in Metal-Catecholates as a Novel Active Site for Oxygen Electroreduction.

    PubMed

    Liu, Xuan-He; Hu, Wei-Li; Jiang, Wen-Jie; Yang, Ya-Wen; Niu, Shuai; Sun, Bing; Wu, Jing; Hu, Jin-Song

    2017-08-30

    Metal-nitrogen coordination sites, M-N x (M = Fe, Co, Ni, etc.), have shown great potential to replace platinum group materials as electrocatalysts for oxygen reduction reaction (ORR). However, the real active site in M-N x is still vague to date due to their complicated structure and composition. It is therefore highly desirable but challenging to develop ORR catalysts with novel and clear active sites, which could meet the needs of comprehensive understanding of structure-function relationships and explore new cost-effective and efficient ORR electrocatalysts. Herein, well-defined M-O 6 coordination in metal-catecholates (M-CATs, M = Ni or Co) is discovered to be catalytically active for ORR via a four-electron-dominated pathway. In view of no pyrolysis involved and unambiguous crystalline structure of M-CATs, the M-O 6 octahedral coordination site with distinct structure is determined as a new type of active site for ORR. These findings extend the scope of metal-nonmetal coordination as an active site for ORR and pave a way for bottom-up design of novel electrocatalysts containing M-O 6 coordination.

  13. Xylem formation can be modeled statistically as a function of primary growth and cambium activity.

    PubMed

    Huang, Jian-Guo; Deslauriers, Annie; Rossi, Sergio

    2014-08-01

    Primary (budburst, foliage and shoot) growth and secondary (cambium and xylem) growth of plants play a vital role in sequestering atmospheric carbon. However, their potential relationships have never been mathematically quantified and the underlying physiological mechanisms are unclear. We monitored primary and secondary growth in Picea mariana and Abies balsamea on a weekly basis from 2010 to 2013 at four sites over an altitudinal gradient (25-900 m) in the eastern Canadian boreal forest. We determined the timings of onset and termination through the fitted functions and their first derivative. We quantified the potential relationships between primary growth and secondary growth using the mixed-effects model. We found that xylem formation of boreal conifers can be modeled as a function of cambium activity, bud phenology, and shoot and needle growth, as well as species- and site-specific factors. Our model reveals that there may be an optimal mechanism to simultaneously allocate the photosynthetic products and stored nonstructural carbon to growth of different organs at different times in the growing season. This mathematical link can bridge phenological modeling, forest ecosystem productivity and carbon cycle modeling, which will certainly contribute to an improved prediction of ecosystem productivity and carbon equilibrium. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  14. The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells

    PubMed Central

    Kozmin, Stanislav G.; Sedletska, Yuliya; Reynaud-Angelin, Anne; Gasparutto, Didier; Sage, Evelyne

    2009-01-01

    It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3–8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells. PMID:19174565

  15. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

  16. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

  17. Probing the Active Surface Sites for CO Reduction on Oxide-Derived Copper Electrocatalysts

    DOE PAGES

    Verdaguer-Casadevall, Arnau; Li, Christina W.; Johansson, Tobias P.; ...

    2015-07-30

    CO electroreduction activity on oxide-derived Cu (OD-Cu) was found to correlate with metastable surface features that bind CO strongly. OD-Cu electrodes prepared by H 2 reduction of Cu 2O precursors reduce CO to acetate and ethanol with nearly 50% Faradaic efficiency at moderate overpotential. Temperature-programmed desorption of CO on OD-Cu revealed the presence of surface sites with strong CO binding that are distinct from the terraces and stepped sites found on polycrystalline Cu foil. After annealing at 350 °C, the surface-area corrected current density for CO reduction is 44-fold lower and the Faradaic efficiency is less than 5%. These changesmore » are accompanied by a reduction in the proportion of strong CO binding sites. Here, we propose that the active sites for CO reduction on OD-Cu surfaces are strong CO binding sites that are supported by grain boundaries. Uncovering these sites is a first step toward understanding the surface chemistry necessary for efficient CO electroreduction.« less

  18. Role of ambient ammonia in particulate ammonium formation at a rural site in the North China Plain

    NASA Astrophysics Data System (ADS)

    Meng, Zhaoyang; Xu, Xiaobin; Lin, Weili; Ge, Baozhu; Xie, Yulin; Song, Bo; Jia, Shihui; Zhang, Rui; Peng, Wei; Wang, Ying; Cheng, Hongbing; Yang, Wen; Zhao, Huarong

    2018-01-01

    The real-time measurements of NH3 and trace gases were conducted, in conjunction with semi-continuous measurements of water-soluble ions in PM2.5 at a rural site in the North China Plain (NCP) from May to September 2013 in order to better understand chemical characteristics of ammonia and the impact of secondary ammonium aerosols on formation in the NCP. Extremely high NH3 and NH4+ concentrations were observed after a precipitation event within 7-10 days following urea application. Elevated NH3 levels coincided with elevated NH4+, indicating that NH3 likely influenced particulate ammonium mass. For the sampling period, the average conversion / oxidation ratios for NH4+ (NHR), SO42- (SOR), and NO3- (NOR) were estimated to be 0.30, 0.64, and 0.24, respectively. The increased NH3 concentrations, mainly from agricultural activities and regional transport, coincided with the prevailing meteorological conditions. The high NH3 level with NHR of about 0.30 indicates that the emission of NH3 in the NCP is much higher than needed for aerosol acid neutralisation, and NH3 plays an important role in the formation of secondary aerosols as a key neutraliser. The hourly data obtained were used to investigate gas-aerosol partitioning characteristics using the thermodynamic equilibrium model ISORROPIA-II. Modelled SO42-, NO3-, and NH3 values agree well with the measurements, while the modelled NH4+ values largely underestimate the measurements. Our observation and modelling results indicate that strong acids in aerosol are completely neutralised. Additional NH4+ exists in aerosol, probably a result of the presence of a substantial amount of oxalic and other diacids.

  19. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Phillips, J.; Warby, C; Whitby, F

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connectedmore » by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.« less

  20. Selective Formation of Ser-His Dipeptide via Phosphorus Activation

    NASA Astrophysics Data System (ADS)

    Shu, Wanyun; Yu, Yongfei; Chen, Su; Yan, Xia; Liu, Yan; Zhao, Yufen

    2018-04-01

    The Ser-His dipeptide is the shortest active peptide. This dipeptide not only hydrolyzes proteins and DNA but also catalyzes the formation of peptides and phosphodiester bonds. As a potential candidate for the prototype of modern hydrolase, Ser-His has attracted increasing attention. To explore if Ser-His could be obtained efficiently in the prebiotic condition, we investigated the reactions of N-DIPP-Ser with His or other amino acids in an aqueous system. We observed that N-DIPP-Ser incubated with His can form Ser-His more efficiently than with other amino acids. A synergistic effect involving the two side chains of Ser and His is presumed to be the critical factor for the selectivity of this specific peptide formation.

  1. Effects of Cola-Flavored Beverages and Caffeine on Streptococcus mutans Biofilm Formation and Metabolic Activity.

    PubMed

    Dotsey, Roger P; Moser, Elizabeth A S; Eckert, George J; Gregory, Richard L

    To examine the effects of cola-flavored beverages and caffeine on growth and metabolism of Streptococcus mutans biofilm. This study was designed to determine if carbonated beverages or caffeine can increase S. mutans growth and biofilm formation and metabolic activity in vitro, potentially leading to increased S. mutans-associated cariogenicity in children that consume them. Six different cola-flavored products, plus pure caffeine, and pure high fructose corn syrup (HFCS), at different concentrations similar to those in the beverages were tested. A 16-hour culture of S. mutans was treated with different dilutions in bacteriological media. To test for the effect on biofilm formation, the biofilm was stained with crystal violet. The absorbance was determined to evaluate biofilm growth. Biofilm metabolic activity was measured based on biofilm having the ability to reduce XTT to a water-soluble orange compound. The inclusion of HFCS in the beverages, as well as pure HFCS, significantly enhanced bacterial biofilm formation and metabolic activity. Pure caffeine and the presence of caffeine in beverages did not significantly increase biofilm formation, but pure caffeine significantly increased metabolism, and Diet Coke had significantly greater metabolic activity than Caffeine-Free Diet Coke. HFCS increases both the biofilm formation and metabolism of S. mutans, and caffeine in some cases increases metabolism of S. mutans.

  2. Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.

    Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

  3. Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

    DOE PAGES

    Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.; ...

    2018-02-05

    Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

  4. Catalytic carbide formation at aluminium-carbon interfaces

    NASA Technical Reports Server (NTRS)

    Maruyama, B.; Rabenberg, L.; Ohuchi, F. S.

    1990-01-01

    X-ray photoelectron spectroscopy investigations of the reaction of several monolayer-thick films of aluminum with glassy carbon substrates are presented. The influence of molecular oxygen and water vapor on the rate of reaction is examined. It is concluded that water vapor catalyzed the formation of aluminum carbide from aluminum and carbon by forming active sites which weakened carbon-carbon bonds at the glassy carbon surface, thus assisting their cleavage. The rate of carbide formation for undosed and molecular oxygen-dosed examples was less as neither metallic aluminum nor oxygen-formed alumina could bond to the carbon atom with sufficient strength to dissociate it quickly.

  5. Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

    PubMed Central

    Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

    2016-01-01

    Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

  6. Rapid Conversion of Traditional Introductory Physics Sequences to an Activity-Based Format

    ERIC Educational Resources Information Center

    Yoder, Garett; Cook, Jerry

    2014-01-01

    The Department of Physics at EKU [Eastern Kentucky University] with support from the National Science Foundations Course Curriculum and Laboratory Improvement Program has successfully converted our entire introductory physics sequence, both algebra-based and calculus-based courses, to an activity-based format where laboratory activities,…

  7. Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

    PubMed

    Mohr, S; Stamler, J S; Brüne, B

    1994-07-18

    Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

  8. Formation and hydrodynamic characteristics of aerobic granules in an activated sludge system.

    PubMed

    Ganesan, M V; Saravanan, V; Sreekrishnan, T R

    2007-02-01

    Development of aerobic granules in the aeration tank of an activated sludge system has been studied. The introduction of activated carbon particles into the aeration tank resulted in the formation of biogranules containing activated carbon as core nuclei. The presence of activated carbon also induced the formation of self-immobilized granules, which did not have any carrier particle at their core. The presence of aerobic granules enhanced the treatment efficiency of the reactor. At an organic loading rate of 32.8 kg COD m(-3)d(-1) and 0.78 h hydraulic retention time (HRT), the reactor showed 96% COD removal efficiency. At an HRT of 0.272 h and organic loading rate of 46.7 kg COD m(-3)d(-1), the reactor outlet COD remained below 100 mg l(-1). Settling velocity studies carried out on the biogranules showed that the drag coefficient of biogranules is greater than that of the rigid particle at the same Reynolds number.

  9. Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

    PubMed

    Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

    2018-05-30

    Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

  10. Identity Agents: Parents as Active and Reflective Participants in their Children's Identity Formation

    ERIC Educational Resources Information Center

    Schachter, Elli P.; Ventura, Jonathan J.

    2008-01-01

    The paper introduces the concept of identity agents. This concept refers to those individuals who actively interact with children and youth with the intention of participating in their identity formation, and who reflectively mediate larger social influences on identity formation. This contrasts with the focus of mainstream research in the…

  11. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

  12. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

    DOE PAGES

    O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

    2016-12-22

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

  13. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Carra,J.; McHugh, C.; Mulligan, S.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding tomore » a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.« less

  14. Monitoring of active layer thermal regime and depth on CALM-S site, James Ross Island, Eastern Antarctic Peninsula

    NASA Astrophysics Data System (ADS)

    Hrbáček, Filip; Kňažková, Michaela; Nývlt, Daniel; Láska, Kamil; Mueller, Carsten W.; Ondruch, Jakub

    2017-04-01

    Active layer thickness and its dynamic are considered one of the key parameters of permafrost-affected ground. They variability are very sensitive to specific local conditions, especially climate, vegetation, snow cover or soil texture and moisture. To better understand the local variability of active layer thickness in Antarctica, the original Circumpolar Active Layer Monitoring protocol (CALM) was adapted as its southern form (CALM-S) with respect to specific conditions of Antarctica. To date, almost 40 CALM-S sites were registered across the Antarctic continent with the highest density on western Antarctic Peninsula (South Shetlands) and Victoria Land in East Antarctica (McMurdo region). On James Ross Island, CALM-S site was established in February 2014 as the first CALM-S in the eastern Antarctic Peninsula region. The CALM-S site is located near the Johann Gregor Mendel Station on the northern coast of James Ross Island. The area delimited to 80 × 70 m is elevated at 8 to 11 m asl. Geologically it consists of a Holocene marine terrace ( 80% of CALM-S area) with typical sandy material and passes to lithified to poorly disintegrated sedimentary rocks of Cretaceous Whisky Bay Formation ( 20% of CALM-S area) with a more muddy material and a typical bimodal composition. For both geologically different parts of CALM-S site, ground temperature was measured at two profiles at several levels up to 200 cm depth using resistance thermometers Pt100/8 (accuracy ± 0.15 °C). The air temperature at 2 m above surface was monitored at the automatic weather station near Johann Gregor Mendel Station using resistance thermometer Pt100/A (accuracy ± 0.15 °C). Data used in this study were obtained during the period from 1 March 2013 to 6 February 2016. Mechanical probing of active layer depth was performed in 72 grid points at the end of January, or beginning of February in 2014 to 2016. During the whole study period, mean annual air temperature varied between -7.0 °C (2013

  15. Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

    PubMed

    Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

    2016-11-15

    The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

  16. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... DEPARTMENT OF ENERGY Reimbursement for Costs of Remedial Action at Active Uranium and Thorium...) acceptance of claims in FY 2011 from eligible active uranium and thorium processing site licensees for... incurred by licensees at active uranium and thorium processing sites to remediate byproduct material...

  17. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    DOE PAGES

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; ...

    2015-03-25

    5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. Here, we mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation ofmore » altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. In conclusion, the overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.« less

  18. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.

    5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. Here, we mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation ofmore » altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. In conclusion, the overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.« less

  19. Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

    PubMed

    Krajewska, Barbara; Zaborska, Wiesława

    2007-10-01

    In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

  20. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    PubMed Central

    Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  1. Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

    PubMed Central

    Gorres, Kelly L.; Pua, Khian Hong; Raines, Ronald T.

    2009-01-01

    The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change. PMID:19890397

  2. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

  3. Transfer hydrogenation over sodium-modified ceria: Enrichment of redox sites active for alcohol dehydrogenation

    DOE PAGES

    Nelson, Nicholas C.; Boote, Brett W.; Naik, Pranjali; ...

    2017-01-17

    Ceria (CeO 2) and sodium-modified ceria (Ce-Na) were prepared through combustion synthesis. Palladium was deposited onto the supports (Pd/CeO 2 and Pd/Ce-Na) and their activity for the aqueous-phase transfer hydrogenation of phenol using 2-propanol under liquid flow conditions was studied. Pd/Ce-Na showed a marked increase (6×) in transfer hydrogenation activity over Pd/CeO 2. Material characterization indicated that water-stable sodium species were not doped into the ceria lattice, but rather existed as subsurface carbonates. Modification of ceria by sodium provided more adsorption and redox active sites (i.e. defects) for 2-propanol dehydrogenation. This effect was an intrinsic property of the Ce-Na supportmore » and independent of Pd. The redox sites active for 2-propanol dehydrogenation were thermodynamically equivalent on both supports/catalysts. At high phenol concentrations, the reaction was limited by 2-propanol adsorption. Furthermore, the difference in catalytic activity was attributed to the different numbers of 2-propanol adsorption and redox active sites on each catalyst.« less

  4. Improved ethanol electrooxidation performance by shortening Pd-Ni active site distance in Pd-Ni-P nanocatalysts

    NASA Astrophysics Data System (ADS)

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-01

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (~5 nm) Pd-Ni-P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni-P heterodimers into Pd-Ni-P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.

  5. Near-IR MCD of the nonheme ferrous active site in naphthalene 1,2-dioxygenase: correlation to crystallography and structural insight into the mechanism of Rieske dioxygenases.

    PubMed

    Ohta, Takehiro; Chakrabarty, Sarmistha; Lipscomb, John D; Solomon, Edward I

    2008-02-06

    Near-IR MCD and variable temperature, variable field (VTVH) MCD have been applied to naphthalene 1,2-dioxygenase (NDO) to describe the coordination geometry and electronic structure of the mononuclear nonheme ferrous catalytic site in the resting and substrate-bound forms with the Rieske 2Fe2S cluster oxidized and reduced. The structural results are correlated with the crystallographic studies of NDO and other related Rieske nonheme iron oxygenases to develop molecular level insights into the structure/function correlation for this class of enzymes. The MCD data for resting NDO with the Rieske center oxidized indicate the presence of a six-coordinate high-spin ferrous site with a weak axial ligand which becomes more tightly coordinated when the Rieske center is reduced. Binding of naphthalene to resting NDO (Rieske oxidized and reduced) converts the six-coordinate sites into five-coordinate (5c) sites with elimination of a water ligand. In the Rieske oxidized form the 5c sites are square pyramidal but transform to a 1:2 mixture of trigonal bipyramial/square pyramidal sites when the Rieske center is reduced. Thus the geometric and electronic structure of the catalytic site in the presence of substrate can be significantly affected by the redox state of the Rieske center. The catalytic ferrous site is primed for the O2 reaction when substrate is bound in the active site in the presence of the reduced Rieske site. These structural changes ensure that two electrons and the substrate are present before the binding and activation of O2, which avoids the uncontrolled formation and release of reactive oxygen species.

  6. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  7. Formation and removal of genotoxic activity during UV/H(2)O(2)-GAC treatment of drinking water.

    PubMed

    Heringa, M B; Harmsen, D J H; Beerendonk, E F; Reus, A A; Krul, C A M; Metz, D H; Ijpelaar, G F

    2011-01-01

    The objective of this study was to determine the genotoxic activity of water after UV/H(2)O(2) oxidation and GAC filtration. Pre-treated surface water from three locations was treated with UV/H(2)O(2) with medium pressure (MP) lamps and passed through granulated activated carbon (GAC). Samples taken before and after each treatment step were extracted and concentrated by solid phase extraction (SPE) and analyzed for genotoxicity using the Comet assay with HepG2 cells and the Ames II assay. The Comet assay showed no genotoxic response in any of the samples. In the Ames II, no genotoxic response was obtained with the TAMix (a mix of six strains), but the TA98 strain showed an increase in genotoxic activity after MP-UV/H(2)O(2) for all three locations. GAC post treatment effectively reduced the activities to control levels at two of the three locations and to below the level of the pre-treated water at one site. The results indicate that UV/H(2)O(2) treatment may lead to the formation of genotoxic by-products, which can be removed by subsequent GAC filtration. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. Studies of star formation in isolated small dark clouds - II. A southern ammonia survey

    NASA Astrophysics Data System (ADS)

    Bourke, T. L.; Hyland, A. R.; Robinson, G.; James, S. D.; Wright, C. M.

    1995-10-01

    A study of the set of small, southern molecular clouds (globules) compiled by Bourke, Hyland & Robinson has been undertaken, through radio observations of ammonia using the Parkes 64-m telescope. The aim of the study is to determine the physical characteristics of the globules, their role in the formation of low-mass stars, and the physical mechanism that triggers the star formation process, or stabilizes the globules against collapse. With this general aim in mind, the (1,1) and (2,2) inversion transitions of ammonia have been surveyed in order to determine the densities, temperatures and masses of the globules. Half of the globules have been detected in ammonia, but only 6 per cent of the detections are `strong' (T*_a>=0.35K). Comparing the globule properties with those of Benson & Myers for cores within complexes, we find that the globules are less opaque and less dense, and are less active sites of star formation. Other properties are comparable. The Vela cometary globules are detected more readily in ammonia than the more isolated globules, and are more active star formation sites. These results suggest that the dense core's environment, in particular the presence of either a large external mass or a significant stellar wind, plays an important role in initiating the star formation process.

  9. Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

    PubMed Central

    Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

    2011-01-01

    Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

  10. A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

    PubMed

    Jacewicz, Agata; Trzemecka, Anna; Guja, Kip E; Plochocka, Danuta; Yakubovskaya, Elena; Bebenek, Anna; Garcia-Diaz, Miguel

    2013-01-01

    Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

  11. Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

    PubMed

    Ibrahim, Firas; Andre, Claire; Iutzeler, Anne; Guillaume, Yves Claude

    2013-10-01

    A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k(cat)) was increased while the Michaelis constant (K(m)) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.

  12. The H(2) sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site.

    PubMed

    Buhrke, Thorsten; Brecht, Marc; Lubitz, Wolfgang; Friedrich, Bärbel

    2002-09-01

    [NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

  13. 40Ar/(39)Ar dating of Chemeron Formation strata encompassing the site of hominid KNM-BC 1, Tugen Hills, Kenya.

    PubMed

    Deino, Alan L; Hill, Andrew

    2002-01-01

    A fossil hominid temporal bone (KNM-BC 1) from surface exposures at Baringo Paleontological Research Project site BPRP#2 in the Chemeron Formation outcropping in a tributary drainage of the Kapthurin River west of Lake Baringo, Kenya has been attributed to Homo sp. indet. K-feldspar phenocrysts from lapilli tuffs bracketing the inferred fossiliferous horizon yield single-crystal(40)Ar/(39)Ar ages of 2.456+/-0.006 and 2.393+/-0.013 Ma. These age determinations are supported by stratigraphically consistent ages on higher tuff horizons and from nearby sections. In addition, new(40)Ar/(39)Ar ages on tuffaceous units near the base and top of the formation along the Kapthurin River yield 3.19+/-0.03 and 1.60+/-0.05 Ma respectively. The base of the formation along the Kapthurin River is thus approximately 0.5 Ma younger than the uppermost Chemeron Formation strata exposed at Tabarin, 23 km to the north-northwest. The upper half of the formation along the Kapthurin River was deposited at an average rate of approximately 11 cm/ka, compared to 21-23 cm/ka at Tabarin. Copyright 2002 Academic Press.

  14. Work-site wellness programmes in Sweden: a cross-sectional study of physical activity, self-efficacy, and health.

    PubMed

    Gånedahl, H; Zsaludek Viklund, P; Carlén, K; Kylberg, E; Ekberg, J

    2015-05-01

    In Sweden, a work-site wellness programme implies reimbursing some of the expenses for health-promoting activities. Although work-site wellness programmes are readily available in Sweden, a large number of employees elect not to participate. The aim of this study was to investigate the association of physical activity, self-reported general health assessment and self-efficacy with participation in a work-site wellness programme. A cross-sectional study design was used. An online questionnaire was distributed to employees of a manufacturing company with 2500 employees in southwest Sweden. Those who took advantage of the work-site wellness programme assessed their general health as better and had higher assessment of physical activity. The study showed that being enlisted also implies a higher level of physical activity and general health; however, the effect sizes of these correlations were small. Self-efficacy, i.e. perceived behavioural control, was not associated with participation in the work-site wellness programme. However, self-efficacy was correlated with both general health assessment and physical activity. A regression analysis to determine explanatory contributions to the general health assessment score showed no significant contribution from participation in a work-site wellness programme, but was instead explained by perceived behavioural control and physical activity. Given the small effect size of the difference in physical activity between participators and non-participators in the work-site wellness programme, it is probable that only a small proportion of participators changed their health-promoting activities as a result of the work-site wellness programme. Copyright © 2015 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

  15. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    DOE PAGES

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH boundmore » structures have the highest calculated activity to date.« less

  16. Magnetic suppression of turbulence and the star formation activity of molecular clouds

    NASA Astrophysics Data System (ADS)

    Zamora-Avilés, Manuel; Vázquez-Semadeni, Enrique; Körtgen, Bastian; Banerjee, Robi; Hartmann, Lee

    2018-03-01

    We present magnetohydrodynamic simulations aimed at studying the effect of the magnetic suppression of turbulence (generated through various instabilities during the formation of molecular clouds by converging) on the subsequent star formation (SF) activity. We study four magnetically supercritical models with magnetic field strengths B = 0, 1, 2, and 3 μG (corresponding to mass-to-flux ratios of ∞, 4.76, 2.38, and 1.59 times the critical value), with the magnetic field, initially being aligned with the flows. We find that, for increasing magnetic field strength, the clouds formed tend to be more massive, denser, less turbulent, and with higher SF activity. This causes the onset of SF activity in the non-magnetic or more weakly magnetized cases to be delayed by a few Myr in comparison to the more strongly magnetized cases. We attribute this behaviour to the suppression of the non-linear thin shell instability (NTSI) by the magnetic field, previously found by Heitsch and coworkers. This result is contrary to the standard notion that the magnetic field provides support to the clouds, thus reducing their star formation rate. However, our result is a completely non-linear one, and could not be foreseen from simple linear considerations.

  17. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

    PubMed

    Kumar, Raj; Calhoun, William J

    2008-12-01

    Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and

  18. THE FORMATION AND MAGNETIC STRUCTURES OF ACTIVE-REGION FILAMENTS OBSERVED BY NVST, SDO, AND HINODE

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yan, X. L.; Xue, Z. K.; Wang, J. C.

    2015-08-15

    To better understand the properties of solar active-region filaments, we present a detailed study on the formation and magnetic structures of two active-region filaments in active region NOAA 11884 during a period of four days. It is found that the shearing motion of the opposite magnetic polarities and the rotation of the small sunspots with negative polarity play an important role in the formation of two active-region filaments. During the formation of these two active-region filaments, one foot of the filaments was rooted in a small sunspot with negative polarity. The small sunspot rotated not only around another small sunspotmore » with negative polarity, but also around the center of its umbra. By analyzing the nonlinear force-free field extrapolation using the vector magnetic fields in the photosphere, twisted structures were found in the two active-region filaments prior to their eruptions. These results imply that the magnetic fields were dragged by the shearing motion between opposite magnetic polarities and became more horizontal. The sunspot rotation twisted the horizontal magnetic fields and finally formed the twisted active-region filaments.« less

  19. MX Siting Investigation. Water Resources Program Industry Activity Inventory, Nevada-Utah.

    DTIC Science & Technology

    1980-09-02

    sites. New and revived mining activities and the cooling needs of possible new coal -fired electric power plants represent the chief competitors with MX...34 !- ---- ON CO. Figure .-. Ma showing araipce yUaIoto f𔃻XMsieCmlx 1 3 include new mining activity and coal -fired, geothermal, and hydroelectric j energy...in northeastern Juab County. The Soil Conservation Service has been actively pushing land treatment programs to increase the productivity of irrigated

  20. Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements--method, rationale, and results on the activation energy of dioxygen formation.

    PubMed

    Buchta, Joachim; Grabolle, Markus; Dau, Holger

    2007-06-01

    The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 mus to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 mus (at 20 degrees C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 degrees C, pH 6.4). In the second part of the study, the temperature dependence (-2.7 to 27.5 degrees C) of the rate constant of dioxygen formation (600/s at 20 degrees C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.

  1. Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sullivan, Sarah M.; Holyoak, Todd

    2008-09-17

    The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics andmore » catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.« less

  2. Enzymes With Lid-Gated Active Sites Must Operate By An Induced Fit Mechanism Instead of Conformational Selection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sullivan, S.M.; Holyoak, T.

    2009-05-26

    The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics andmore » catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.« less

  3. Enthalpic Breakdown of Water Structure on Protein Active-Site Surfaces

    PubMed Central

    Haider, Kamran; Wickstrom, Lauren; Ramsey, Steven; Gilson, Michael K.; Kurtzman, Tom

    2016-01-01

    The principles underlying water reorganization around simple non-polar solutes are well understood and provide the framework for classical hydrophobic effect, whereby water molecules structure themselves around solutes so that they maintain favorable energetic contacts with both the solute and with other water molecules. However, for certain solute surface topographies, water molecules, due to their geometry and size, are unable to simultaneously maintain favorable energetic contacts with both the surface and neighboring water molecules. In this study, we analyze the solvation of ligand-binding sites for six structurally diverse proteins using hydration site analysis and measures of local water structure, in order to identify surfaces at which water molecules are unable to structure themselves in a way that maintains favorable enthalpy relative to bulk water. These surfaces are characterized by a high degree of enclosure, weak solute-water interactions, and surface constraints that induce unfavorable pair interactions between neighboring water molecules. Additionally, we find that the solvation of charged side-chains in an active site generally results in favorable enthalpy but can also lead to pair interactions between neighboring water molecules that are significantly unfavorable relative to bulk water. We find that frustrated local structure can occur not only in apolar and weakly polar pockets, where overall enthalpy tends to be unfavorable, but also in charged pockets, where overall water enthalpy tends to be favorable. The characterization of local water structure in these terms may prove useful for evaluating the displacement of water from diverse protein active-site environments. PMID:27169482

  4. Nevada National Security Site Industrial Sites Project Closeout - 12498

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cabble, Kevin; Krauss, Mark; Matthews, Pat

    The U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office is responsible for environmental restoration (ER) at the Nevada National Security Site (NNSS). This includes remediation at Industrial Sites where past nuclear testing activities and activities that supported nuclear testing may have or are known to have resulted in the release of contaminants into the environment. Industrial Sites at the NNSS have included nuclear facilities that supported the nuclear rocket/missile development programs, gas stations, landfills, spill sites, ordnance sites, and numerous other waste disposal and release sites. The NNSS Industrial Sites activities neared completion at the endmore » of fiscal year 2011 while other activities required under the Federal Facility Agreement and Consent Order (FFACO) and part of the same NNSS ER Project are forecasted to extend to 2027 or beyond. With the majority of Industrial Sites corrective action units (CAUs) completed (more than 250 CAUs and over 1,800 corrective action sites), it was determined that an activity closeout process should be implemented to ensure that the work completed over the past 15 years is well documented in a comprehensive and concise summary. While the process used to close each individual CAU is described in approved documents, no single document describes in summary fashion the work completed to close the many individual Industrial Sites. The activity closeout process will be used to develop an Industrial Sites closeout document that describes these years of work. This document will summarize the number of Industrial Sites closed under the FFACO and provide general descriptions of projects, contaminants removed, and sites closed in place with corresponding Use Restrictions. Other pertinent information related to Industrial Sites work such as the project history, closure decisions, historical declarations, remediation strategies, and final CAU status will be included in the

  5. Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ.

    PubMed

    Chertkov, O V; Armeev, G A; Uporov, I V; Legotsky, S A; Sykilinda, N N; Shaytan, A K; Klyachko, N L; Miroshnikov, K A

    2017-01-01

    Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C -terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

  6. Influence of a Confined Methanol Solvent on the Reactivity of Active Sites in UiO-66.

    PubMed

    Caratelli, Chiara; Hajek, Julianna; Rogge, Sven M J; Vandenbrande, Steven; Meijer, Evert Jan; Waroquier, Michel; Van Speybroeck, Veronique

    2018-02-19

    UiO-66, composed of Zr-oxide bricks and terephthalate linkers, is currently one of the most studied metal-organic frameworks due to its exceptional stability. Defects can be introduced in the structure, creating undercoordinated Zr atoms which are Lewis acid sites. Here, additional Brønsted sites can be generated by coordinated protic species from the solvent. In this Article, a multilevel modeling approach was applied to unravel the effect of a confined methanol solvent on the active sites in UiO-66. First, active sites were explored with static periodic density functional theory calculations to investigate adsorption of water and methanol. Solvent was then introduced in the pores with grand canonical Monte Carlo simulations, followed by a series of molecular dynamics simulations at operating conditions. A hydrogen-bonded network of methanol molecules is formed, allowing the protons to shuttle between solvent methanol, adsorbed water, and the inorganic brick. Upon deprotonation of an active site, the methanol solvent aids the transfer of protons and stabilizes charged configurations via hydrogen bonding, which could be crucial in stabilizing reactive intermediates. The multilevel modeling approach adopted here sheds light on the important role of a confined solvent on the active sites in the UiO-66 material, introducing dynamic acidity in the system at finite temperatures by which protons may be easily shuttled from various positions at the active sites. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  7. Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

    PubMed Central

    Aoyama, M; Grabowski, D R; Dubyak, G R; Constantinou, A I; Rybicki, L A; Bukowski, R M; Ganapathi, M K; Hickson, I D; Ganapathi, R

    1998-01-01

    Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients

  8. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

    PubMed

    de San Martin, Javier Zorrilla; Jalil, Abdelali; Trigo, Federico F

    2015-12-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca(2+) photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl(-)](i), autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30-150 GABA(A) channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na(v)-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. © 2015 Zorrilla de San Martin et al.

  9. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  10. Recent Experience Using Active Love Wave Techniques to Characterize Seismographic Station Sites

    NASA Astrophysics Data System (ADS)

    Martin, A. J.; Yong, A.; Salomone, L.

    2014-12-01

    Active-source Love waves recorded by the multi-channel analysis of surface wave (MASLW) technique were recently analyzed in two site characterization projects. Between 2010 and 2011, the 2009 American Recovery and Reinvestment Act (ARRA) funded GEOVision to conduct geophysical investigations at 189 seismographic stations—185 in California and 4 in the Central Eastern U.S. (CEUS). The original project plan was to utilize active and passive Rayleigh wave-based techniques to obtain shear-wave velocity (VS) profiles to a minimum depth of 30 m and the time-averaged VS of the upper 30 meters (VS30). Early in the investigation it became evident that Rayleigh wave techniques, such as multi-channel analysis of surface waves (MASRW), were not effective at characterizing all sites. Shear-wave seismic refraction and MASLW techniques were therefore applied. The MASLW technique was deployed at a total of 38 sites, in addition to other methods, and used as the primary technique to characterize 22 sites, 5 of which were also characterized using Rayleigh wave techniques. In 2012, the Electric Power Research Institute funded characterization of 33 CEUS station sites. Based on experience from the ARRA investigation, both MASRW and MASLW data were acquired by GEOVision at 24 CEUS sites—the remaining 9 sites and 2 overlapping sites were characterized by University of Texas, Austin. Of the 24 sites characterized by GEOVision, 16 were characterized using MASLW data, 4 using both MASLW and MASRW data and 4 using MASRW data. Love wave techniques were often found to perform better, or at least yield phase velocity data that could be more readily modeled using the fundamental mode assumption, at shallow rock sites, sites with steep velocity gradients, and, sites with a thin, low velocity, surficial soil layer overlying stiffer sediments. These types of velocity structure often excite dominant higher modes in Rayleigh wave data, but not in Love wave data. At such sites, it may be possible

  11. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    PubMed

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC 50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - C ata L ytic A ctive S ite P rediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro

  13. The role of the peripheral benzodiazepine receptor in photodynamic activity of certain pyropheophorbide ether photosensitizers: albumin site II as a surrogate marker for activity.

    PubMed

    Dougherty, Thomas J; Sumlin, Adam B; Greco, William R; Weishaupt, Kenneth R; Vaughan, Lurine A; Pandey, Ravindra K

    2002-07-01

    A study has been carried out to define the importance of the peripheral benzodiazepine receptor (PBR) as a binding site for a series of chlorin-type photosensitizers, pyropheophorbide-a ethers, the subject of a previous quantitative structure-activity relationship study by us. The effects of the PBR ligand PK11195 on the photodynamic activity have been determined in vivo for certain members of this series of alkyl-substituted ethers: two of the most active derivatives (hexyl and heptyl), the least active derivative (dodecyl [C12]) and one of intermediate activity (octyl [C8]). The photodynamic therapy (PDT) effect was inhibited by PK11195 for both of the most active derivatives, but no effect on PDT activity was found for the less active C12 or C8 ethers. The inhibitory effects of PK11195 were predicted by the binding of only the active derivatives to the benzodiazepine site on albumin, ie. human serum albumin (HSA)-Site II. Thus, as with certain other types of photosensitizers, it has been demonstrated with this series of pyropheophorbide ethers that in vitro binding to HSA-Site II is a predictor of both optimal in vivo activity and binding to the PBR in vivo.

  14. In Vitro Effects of Sports and Energy Drinks on Streptococcus mutans Biofilm Formation and Metabolic Activity.

    PubMed

    Vinson, LaQuia A; Goodlett, Amy K; Huang, Ruijie; Eckert, George J; Gregory, Richard L

    2017-09-15

    Sports and energy drinks are being increasingly consumed and contain large amounts of sugars, which are known to increase Streptococcus mutans biofilm formation and metabolic activity. The purpose of this in vitro study was to investigate the effects of sports and energy drinks on S. mutans biofilm formation and metabolic activity. S. mutans UA159 was cultured with and without a dilution (1:3 ratio) of a variety of sports and energy drinks in bacterial media for 24 hours. The biofilm was washed, fixed, and stained. Biofilm growth was evaluated by reading absorbance of the crystal violet. Biofilm metabolic activity was measured by the biofilm-reducing XTT to a water-soluble orange compound. Gatorade Protein Recovery Shake and Starbucks Doubleshot Espresso Energy were found to significantly increase biofilm (30-fold and 22-fold, respectively) and metabolic activity (2-fold and 3-fold, respectively). However, most of the remaining drinks significantly inhibited biofilm growth and metabolic activity. Several sports and energy drinks, with sugars or sugar substitutes as their main ingredients inhibited S. mutans biofilm formation. Among the drinks evaluated, Gatorade Protein Recovery Chocolate Shake and Starbucks Doubleshot Energy appear to have cariogenic potential since they increased the biofilm formation and metabolic activity of S. mutans.

  15. Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

    2010-07-20

    Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

  16. Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

    PubMed Central

    Deng, Hua

    2017-01-01

    Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

  17. Improved ethanol electrooxidation performance by shortening Pd–Ni active site distance in Pd–Ni–P nanocatalysts

    PubMed Central

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-01

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (∼5 nm) Pd–Ni–P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni–P heterodimers into Pd–Ni–P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites. PMID:28071650

  18. Dithiolato-bridged nickel-iron complexes as models for the active site of [NiFe]-hydrogenases.

    PubMed

    Song, Li-Cheng; Yang, Xi-Yue; Cao, Meng; Gao, Xiu-Yun; Liu, Bei-Bei; Zhu, Liang; Jiang, Feng

    2017-03-30

    The structural and functional modeling of the active site of [NiFe]-hydrogenases has been proved to be challenging to a great extent. Herein, we report the synthesis, structures, and some properties of the NiFe-based dicarbonyl, terminal hydride, and μ-hydroxo models for the active site of [NiFe]-hydrogenases.

  19. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

    PubMed

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  20. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model

    PubMed Central

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  1. Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*

    PubMed Central

    Zhang, Shengnan; Fitzpatrick, Paul F.

    2016-01-01

    Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25–117) is the regulatory domain of PheH lacking residues 1–24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25–117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25–117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. PMID:26823465

  2. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  3. A qualitative examination of perceptions of physical activity guidelines and preferences for format.

    PubMed

    Berry, Tanya R; Witcher, Chad; Holt, Nicholas L; Plotnikoff, Ronald C

    2010-11-01

    A descriptive exploratory study was conducted to gain an understanding of public perceptions of physical activity guidelines and to discover what formats appeal to participants. Canada's Physical Activity Guide (CPAG) was used as an example of such guidelines. Data were collected from 22 participants in five focus groups (composed of female undergraduate students, female office workers, male office workers, participants in a Type II diabetes rehabilitation program, and participants in a cardiovascular rehabilitation program). Cross-case qualitative analyses were conducted. Six themes emerged under the general categories of familiarity and preferences for PA promotional materials. In terms of familiarity, participants lacked awareness of CPAG and criticized its format. In terms of preferences, participants encouraged the use of stylistically similar messaging to those used by commercial advertisers, wanted personal stories, Internet-based media, and the use of celebrities' success stories. There was little awareness of CPAG and the current format was unappealing.

  4. The Formation and Development of Cognitive Activity of Students in the Learning Process

    ERIC Educational Resources Information Center

    Saparkyzy, Zhannat; Isatayeva, Gulzhan; Kozhabekova, Zahida; Zhakesheva, Aimzhan; Koptayeva, Gulzhamal; Agabekova, Gulzhan; Agabekova, Sholpan

    2016-01-01

    In this article we will discuss how the holding of a special and dedicated work helped to change the levels of formation of the major components of cognitive activity. Cognitive activity with the content aspect is a system of perceptual, mnemonic and intellectual activity and from the form--as an individual, joint, or pseudo-individual pseudo…

  5. Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

    PubMed Central

    Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

    2008-01-01

    SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

  6. Quenching of the Star Formation Activity of Galaxies in Dense Environments

    NASA Astrophysics Data System (ADS)

    Boselli, A.

    2017-12-01

    The nearby Universe is an ideal laboratory to study the effects of the environments on galaxy evolution. We have analysed the multifrequency properties of galaxies in the nearby clusters Virgo, Coma, and A1367. We have shown that the HI gas content and the activity of star formation of the late-type galaxies start to gradually decrease inwards ˜ one virial radius. We have also shown that late-type galaxies in these clusters have truncated HI, H_2, dust, and star forming discs once the HI gas content is removed by the harsh environment. Some of these galaxies also exibit spectacular tails of atomic neutral, ionised, or hot gas without any counterpart in the stellar component. All this evidence favors ram pressure stripping as the dominant mechanism responsible for the gas removal from the disc, and for the following quenching of the star formation activity.

  7. Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

    PubMed Central

    Hess, Katharina; Ajjan, Ramzi; Phoenix, Fladia; Dobó, József; Gál, Péter; Schroeder, Verena

    2012-01-01

    Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation. PMID:22536427

  8. Entrapped Single Tungstate Site in Zeolite for Cooperative Catalysis of Olefin Metathesis with Brønsted Acid Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Pu; Ye, Lin; Sun, Zhenyu

    Industrial olefin metathesis catalysts generally suffer from low reaction rates and require harsh reaction conditions for moderate activities. This is due to their inability to prevent metathesis active sites (MAS) from aggregation and their intrinsic poor adsorption and activation of olefin molecules. Here, isolated tungstate species as single molecular MAS is immobilized inside zeolite pores by Bronsted acid sites (BAS) on the inner surface. It is demonstrated for the first time that unoccupied BAS in atomic proximity to MAS enhance olefin adsorption and greatly facilitate the formation of metallocycle intermediates in a stereospecific manner. Thus, effective cooperative catalysis takes placemore » over the BAS-MAS pair. In consequence, for the cross-metathesis of ethene and trans-2-butene to propene, under the same mild reaction conditions, the propene production rate over WOx/USY is ca. 7,300 times that over the industrial WO3/SiO2 based catalyst. A propene yield up to 79% (80% selectivity) without observable deactivation was obtained over WOx/USY for a wide range of reaction conditions.« less

  9. How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

    PubMed Central

    Hytönen, Vesa P; Vogel, Viola

    2008-01-01

    Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

  10. Identification of the fatty acid activation site on human ClC-2.

    PubMed

    Cuppoletti, John; Tewari, Kirti P; Chakrabarti, Jayati; Malinowska, Danuta H

    2017-06-01

    Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl - channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET 691 , of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP] i ), EP 2 , or EP 4 receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl - current activation (increasing EC 50 s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA 655 /RGEA 691 ) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP] i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl - currents without increasing [cAMP] i, while PGE 2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP 2 or EP 4 receptors. L-161,982, a supposed EP 4 -selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl - currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET 691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP] i , and EP 2 or EP 4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2. Copyright © 2017 the American Physiological Society.

  11. Computational active site analysis of molecular pathways to improve functional classification of enzymes.

    PubMed

    Ozyurt, A Sinem; Selby, Thomas L

    2008-07-01

    This study describes a method to computationally assess the function of homologous enzymes through small molecule binding interaction energy. Three experimentally determined X-ray structures and four enzyme models from ornithine cyclo-deaminase, alanine dehydrogenase, and mu-crystallin were used in combination with nine small molecules to derive a function score (FS) for each enzyme-model combination. While energy values varied for a single molecule-enzyme combination due to differences in the active sites, we observe that the binding energies for the entire pathway were proportional for each set of small molecules investigated. This proportionality of energies for a reaction pathway appears to be dependent on the amino acids in the active site and their direct interactions with the small molecules, which allows a function score (FS) to be calculated to assess the specificity of each enzyme. Potential of mean force (PMF) calculations were used to obtain the energies, and the resulting FS values demonstrate that a measurement of function may be obtained using differences between these PMF values. Additionally, limitations of this method are discussed based on: (a) larger substrates with significant conformational flexibility; (b) low homology enzymes; and (c) open active sites. This method should be useful in accurately predicting specificity for single enzymes that have multiple steps in their reactions and in high throughput computational methods to accurately annotate uncharacterized proteins based on active site interaction analysis. 2008 Wiley-Liss, Inc.

  12. Elucidating Oxygen Reduction Active Sites in Pyrolyzed Metal–Nitrogen Coordinated Non-Precious-Metal Electrocatalyst Systems

    PubMed Central

    2015-01-01

    Detailed understanding of the nature of the active centers in non-precious-metal-based electrocatalyst, and their role in oxygen reduction reaction (ORR) mechanistic pathways will have a profound effect on successful commercialization of emission-free energy devices such as fuel cells. Recently, using pyrolyzed model structures of iron porphyrins, we have demonstrated that a covalent integration of the Fe–Nx sites into π-conjugated carbon basal plane modifies electron donating/withdrawing capability of the carbonaceous ligand, consequently improving ORR activity. Here, we employ a combination of in situ X-ray spectroscopy and electrochemical methods to identify the various structural and functional forms of the active centers in non-heme Fe/N/C catalysts. Both methods corroboratively confirm the single site 2e– × 2e– mechanism in alkaline media on the primary Fe2+–N4 centers and the dual-site 2e– × 2e– mechanism in acid media with the significant role of the surface bound coexisting Fe/FexOy nanoparticles (NPs) as the secondary active sites. PMID:24817921

  13. Elucidating Oxygen Reduction Active Sites in Pyrolyzed Metal-Nitrogen Coordinated Non-Precious-Metal Electrocatalyst Systems.

    PubMed

    Tylus, Urszula; Jia, Qingying; Strickland, Kara; Ramaswamy, Nagappan; Serov, Alexey; Atanassov, Plamen; Mukerjee, Sanjeev

    2014-05-01

    Detailed understanding of the nature of the active centers in non-precious-metal-based electrocatalyst, and their role in oxygen reduction reaction (ORR) mechanistic pathways will have a profound effect on successful commercialization of emission-free energy devices such as fuel cells. Recently, using pyrolyzed model structures of iron porphyrins, we have demonstrated that a covalent integration of the Fe-N x sites into π-conjugated carbon basal plane modifies electron donating/withdrawing capability of the carbonaceous ligand, consequently improving ORR activity. Here, we employ a combination of in situ X-ray spectroscopy and electrochemical methods to identify the various structural and functional forms of the active centers in non-heme Fe/N/C catalysts. Both methods corroboratively confirm the single site 2e - × 2e - mechanism in alkaline media on the primary Fe 2+ -N 4 centers and the dual-site 2e - × 2e - mechanism in acid media with the significant role of the surface bound coexisting Fe/Fe x O y nanoparticles (NPs) as the secondary active sites.

  14. Co-doping of (Bi(0.5)Na(0.5))TiO(3): secondary phase formation and lattice site preference of Co.

    PubMed

    Schmitt, V; Staab, T E M

    2012-11-14

    Bismuth sodium titanate (Bi(0.5)Na(0.5))TiO(3) (BNT) is considered to be one of the most promising lead-free alternatives to piezoelectric lead zirconate titanate (PZT). However, the effect of dopants on the material has so far received little attention from an atomic point of view. In this study we investigated the effects of cobalt-doping on the formation of additional phases and determined the preferred lattice site of cobalt in BNT. The latter was achieved by comparing the measured x-ray absorption near-edge structure (XANES) spectra to numerically calculated spectra of cobalt on various lattice sites in BNT. (Bi(0.5)Na(0.5))TiO(3) + x mol% Co (x = 0.0, 0.5, 1.0, 2.6) was synthesized via solid state reaction. As revealed by SEM backscattering images, a secondary phase formed in all doped specimens. Using both XRD and SEM-EDX, it was identified as Co(2)TiO(4) for dopant levels >0.5 mol%. In addition, a certain amount of cobalt was incorporated into BNT, as shown by electron probe microanalysis. This amount increased with increasing dopant levels, suggesting that an equilibrium forms together with the secondary phase. The XANES experiments revealed that cobalt occupies the octahedral B-site in the BNT perovskite lattice, substituting Ti and promoting the formation of oxygen vacancies in the material.

  15. Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

    PubMed Central

    van Weering, David H. J.; de Rooij, Johan; Marte, Barbara; Downward, Julian; Bos, Johannes L.; Burgering, Boudewijn M. T.

    1998-01-01

    Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events. PMID:9528752

  16. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

    2013-07-01

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an abovegroundmore » structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)« less

  17. Greenhouse Gas Fluxes at the Tablelands, NL, Canada: A Site of Active Serpentinization

    NASA Astrophysics Data System (ADS)

    Morrill, P. L.; Morrissey, L. S.; Cumming, E.

    2016-12-01

    Active sites of serpentinization have been proposed as sites for carbon capture and storage (CCS) projects. However, in addition to their ability to convert carbon dioxide to carbonate rock, sites of serpentinization also have the potential release methane, which is a more power greenhouse gas than carbon dioxide. Very little is known about the natural flux of carbon dioxide sequestered and methane released into the atmosphere from active sites of serpentinization. In this study we measured carbon dioxide, methane, and nitrous oxide gas fluxes at a pool of ultra-basic water discharging from serpentinized rock in Winterhouse Canyon, Gros Morne, Newfoundland. We found that the flux of methane released was 4.6 x 10-7 mol/m2/min and the carbon dioxide sequestered was 1.9 x 10-5 mol/m2/min, while the concentrations of nitrous oxide showed little change. Based on these fluxes we calculated predictive climate change parameters such as net radiative forcing and global warming potential which predicted that despite the methane being released the site still had an overall long-term atmospheric cooling effect based on the natural rate of carbon dioxide sequestration.

  18. Generalized trends in the formation energies of perovskite oxides.

    PubMed

    Zeng, ZhenHua; Calle-Vallejo, Federico; Mogensen, Mogens B; Rossmeisl, Jan

    2013-05-28

    Generalized trends in the formation energies of several families of perovskite oxides (ABO3) and plausible explanations to their existence are provided in this study through a combination of DFT calculations, solid-state physics analyses and simple physical/chemical descriptors. The studied elements at the A site of perovskites comprise rare-earth, alkaline-earth and alkaline metals, whereas 3d and 5d metals were studied at the B site. We also include ReO3-type compounds, which have the same crystal structure of cubic ABO3 perovskites except without A-site elements. From the observations we extract the following four conclusions for the perovskites studied in the present paper: for a given cation at the B site, (I) perovskites with cations of identical oxidation state at the A site possess close formation energies; and (II) perovskites with cations of different oxidation states at the A site usually have quite different but ordered formation energies. On the other hand, for a given A-site cation, (III) the formation energies of perovskites vary linearly with respect to the atomic number of the elements at the B site within the same period of the periodic table, and the slopes depend systematically on the oxidation state of the A-site cation; and (IV) the trends in formation energies of perovskites with elements from different periods at the B site depend on the oxidation state of A-site cations. Since the energetics of perovskites is shown to be the superposition of the individual contributions of their constituent oxides, the trends can be rationalized in terms of A-O and B-O interactions in the ionic crystal. These findings reveal the existence of general systematic trends in the formation energies of perovskites and provide further insight into the role of ion-ion interactions in the properties of ternary compounds.

  19. Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site.

    PubMed

    Wang, Lu; Fried, Stephen D; Boxer, Steven G; Markland, Thomas E

    2014-12-30

    Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.

  20. Simplifying healthful choices: a qualitative study of a physical activity based nutrition label format

    PubMed Central

    2013-01-01

    Background This study used focus groups to pilot and evaluate a new nutrition label format and refine the label design. Physical activity equivalent labels present calorie information in terms of the amount of physical activity that would be required to expend the calories in a specified food item. Methods Three focus groups with a total of twenty participants discussed food choices and nutrition labeling. They provided information on comprehension, usability and acceptability of the label. A systematic coding process was used to apply descriptive codes to the data and to identify emerging themes and attitudes. Results Participants in all three groups were able to comprehend the label format. Discussion about label format focused on issues including gender of the depicted figure, physical fitness of the figure, preference for walking or running labels, and preference for information in miles or minutes. Feedback from earlier focus groups was used to refine the labels in an iterative process. Conclusions In contrast to calorie labels, participants shown physical activity labels asked and answered, “How does this label apply to me?” This shift toward personalized understanding may indicate that physical activity labels offer an advantage over currently available nutrition labels. PMID:23742678

  1. Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation.

    PubMed

    Leandro, João; Stokka, Anne J; Teigen, Knut; Andersen, Ole A; Flatmark, Torgeir

    2017-07-01

    Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in l-phenylalanine (l-Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l-Phe is the key player in this transition. X-ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr 138 -loop to the active site in the presence of l-Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr 138 point mutants. A high linear correlation ( r 2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate-activated full-length tetramers. In the tetramers, a correlation ( r 2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l-Phe concentration. The new data support a similar functional importance of the Tyr 138 -loop in the catalytic domain and the full-length enzyme homotetramer.

  2. Inhibitor-based validation of a homology model of the active-site of tripeptidyl peptidase II.

    PubMed

    De Winter, Hans; Breslin, Henry; Miskowski, Tamara; Kavash, Robert; Somers, Marijke

    2005-04-01

    A homology model of the active site region of tripeptidyl peptidase II (TPP II) was constructed based on the crystal structures of four subtilisin-like templates. The resulting model was subsequently validated by judging expectations of the model versus observed activities for a broad set of prepared TPP II inhibitors. The structure-activity relationships observed for the prepared TPP II inhibitors correlated nicely with the structural details of the TPP II active site model, supporting the validity of this model and its usefulness for structure-based drug design and pharmacophore searching experiments.

  3. Reversible active site sulfoxygenation can explain the oxygen tolerance of a NAD+-reducing [NiFe] hydrogenase and its unusual infrared spectroscopic properties.

    PubMed

    Horch, Marius; Lauterbach, Lars; Mroginski, Maria Andrea; Hildebrandt, Peter; Lenz, Oliver; Zebger, Ingo

    2015-02-25

    Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD(+)-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD(+) and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2.

  4. The suppression of star formation by powerful active galactic nuclei.

    PubMed

    Page, M J; Symeonidis, M; Vieira, J D; Altieri, B; Amblard, A; Arumugam, V; Aussel, H; Babbedge, T; Blain, A; Bock, J; Boselli, A; Buat, V; Castro-Rodríguez, N; Cava, A; Chanial, P; Clements, D L; Conley, A; Conversi, L; Cooray, A; Dowell, C D; Dubois, E N; Dunlop, J S; Dwek, E; Dye, S; Eales, S; Elbaz, D; Farrah, D; Fox, M; Franceschini, A; Gear, W; Glenn, J; Griffin, M; Halpern, M; Hatziminaoglou, E; Ibar, E; Isaak, K; Ivison, R J; Lagache, G; Levenson, L; Lu, N; Madden, S; Maffei, B; Mainetti, G; Marchetti, L; Nguyen, H T; O'Halloran, B; Oliver, S J; Omont, A; Panuzzo, P; Papageorgiou, A; Pearson, C P; Pérez-Fournon, I; Pohlen, M; Rawlings, J I; Rigopoulou, D; Riguccini, L; Rizzo, D; Rodighiero, G; Roseboom, I G; Rowan-Robinson, M; Sánchez Portal, M; Schulz, B; Scott, D; Seymour, N; Shupe, D L; Smith, A J; Stevens, J A; Trichas, M; Tugwell, K E; Vaccari, M; Valtchanov, I; Viero, M; Vigroux, L; Wang, L; Ward, R; Wright, G; Xu, C K; Zemcov, M

    2012-05-09

    The old, red stars that constitute the bulges of galaxies, and the massive black holes at their centres, are the relics of a period in cosmic history when galaxies formed stars at remarkable rates and active galactic nuclei (AGN) shone brightly as a result of accretion onto black holes. It is widely suspected, but unproved, that the tight correlation between the mass of the black hole and the mass of the stellar bulge results from the AGN quenching the surrounding star formation as it approaches its peak luminosity. X-rays trace emission from AGN unambiguously, whereas powerful star-forming galaxies are usually dust-obscured and are brightest at infrared and submillimetre wavelengths. Here we report submillimetre and X-ray observations that show that rapid star formation was common in the host galaxies of AGN when the Universe was 2-6 billion years old, but that the most vigorous star formation is not observed around black holes above an X-ray luminosity of 10(44) ergs per second. This suppression of star formation in the host galaxy of a powerful AGN is a key prediction of models in which the AGN drives an outflow, expelling the interstellar medium of its host and transforming the galaxy's properties in a brief period of cosmic time.

  5. The transition state for formation of the peptide bond in the ribosome

    PubMed Central

    Gindulyte, Asta; Bashan, Anat; Agmon, Ilana; Massa, Lou; Yonath, Ada; Karle, Jerome

    2006-01-01

    Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3′ end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, Ea, is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be ≈18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2′ OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments. PMID:16938893

  6. Conserved active site residues limit inhibition of a copper-containing nitrite reductase by small molecules.

    PubMed

    Tocheva, Elitza I; Eltis, Lindsay D; Murphy, Michael E P

    2008-04-15

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  7. Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

    PubMed

    Willey, Christopher D; Balasubramanian, Sundaravadivel; Rodríguez Rosas, María C; Ross, Robert S; Kuppuswamy, Dhandapani

    2003-06-01

    In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.

  8. Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions.

    PubMed

    Ebert, Maximilian C C J C; Morley, Krista L; Volpato, Jordan P; Schmitzer, Andreea R; Pelletier, Joelle N

    2015-04-01

    Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation. © 2014 The Protein Society.

  9. Active Management of Integrated Geothermal-CO2 Storage Reservoirs in Sedimentary Formations

    DOE Data Explorer

    Buscheck, Thomas A.

    2012-01-01

    Active Management of Integrated Geothermal–CO2 Storage Reservoirs in Sedimentary Formations: An Approach to Improve Energy Recovery and Mitigate Risk: FY1 Final Report The purpose of phase 1 is to determine the feasibility of integrating geologic CO2 storage (GCS) with geothermal energy production. Phase 1 includes reservoir analyses to determine injector/producer well schemes that balance the generation of economically useful flow rates at the producers with the need to manage reservoir overpressure to reduce the risks associated with overpressure, such as induced seismicity and CO2 leakage to overlying aquifers. Based on a range of well schemes, techno-economic analyses of the levelized cost of electricity (LCOE) are conducted to determine the economic benefits of integrating GCS with geothermal energy production. In addition to considering CO2 injection, reservoir analyses are conducted for nitrogen (N2) injection to investigate the potential benefits of incorporating N2 injection with integrated geothermal-GCS, as well as the use of N2 injection as a potential pressure-support and working-fluid option. Phase 1 includes preliminary environmental risk assessments of integrated geothermal-GCS, with the focus on managing reservoir overpressure. Phase 1 also includes an economic survey of pipeline costs, which will be applied in Phase 2 to the analysis of CO2 conveyance costs for techno-economics analyses of integrated geothermal-GCS reservoir sites. Phase 1 also includes a geospatial GIS survey of potential integrated geothermal-GCS reservoir sites, which will be used in Phase 2 to conduct sweet-spot analyses that determine where promising geothermal resources are co-located in sedimentary settings conducive to safe CO2 storage, as well as being in adequate proximity to large stationary CO2 sources.

  10. A neurokinin 1 receptor antagonist decreases postoperative peritoneal adhesion formation and increases peritoneal fibrinolytic activity.

    PubMed

    Reed, Karen L; Fruin, A Brent; Gower, Adam C; Stucchi, Arthur F; Leeman, Susan E; Becker, James M

    2004-06-15

    Fibrous adhesions remain a major sequela of abdominal surgery. The proinflammatory peptide substance P (SP), known to participate in inflammatory events, may play a key role in adhesion formation. This hypothesis was tested by using an antagonist, CJ-12,255 (Pfizer), that blocks the binding of SP to the neurokinin 1 receptor (NK-1R). Adhesion formation was surgically induced in the peritoneum of rats receiving daily doses of the NK-1R antagonist (NK-1RA; 5.0 or 10.0 mg/kg per day) or saline. On postoperative day 7, both the low and high doses of NK-1RA significantly (P < 0.05) reduced adhesion formation by 45% and 53%, respectively, compared with controls. Subsequently, the effect of NK-1RA administration on peritoneal fibrinolytic activity was investigated to determine a potential mechanism for SP action in the peritoneum. Samples were collected from nonoperated controls and from animals 24 h postsurgery that were administered either NK-1RA or saline. Fibrinolytic activity in peritoneal fluid was assayed by zymography, and expression of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1, both regulators of fibrinolytic activity, was assessed in peritoneal tissue and fluid by RT-PCR and bioassay, respectively. NK-1RA administration led to a marked (P < 0.05) increase in tPA mRNA levels in peritoneal tissue compared with nonoperated and saline-administered animals. Likewise, NK-1RA administration significantly (P < 0.05) increased tPA in the peritoneal fluid. These data suggest that activation of the NK-1R promotes peritoneal adhesion formation by limiting fibrinolytic activity in the postoperative peritoneum, thus enabling fibrinous adhesions to persist.

  11. LASIC: Light Activated Site-Specific Conjugation of Native IgGs.

    PubMed

    Hui, James Z; Tamsen, Shereen; Song, Yang; Tsourkas, Andrew

    2015-08-19

    Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large.

  12. Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

    PubMed

    Wakasugi, Mitsuo; Sasaki, Takuma; Matsumoto, Megumi; Nagaoka, Miyuki; Inoue, Keiko; Inobe, Manabu; Horibata, Katsuyoshi; Tanaka, Kiyoji; Matsunaga, Tsukasa

    2014-10-10

    Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Formation and spatial distribution of hypervelocity stars in AGN outflows

    NASA Astrophysics Data System (ADS)

    Wang, Xiawei; Loeb, Abraham

    2018-05-01

    We study star formation within outflows driven by active galactic nuclei (AGN) as a new source of hypervelocity stars (HVSs). Recent observations revealed active star formation inside a galactic outflow at a rate of ∼ 15M⊙yr-1 . We verify that the shells swept up by an AGN outflow are capable of cooling and fragmentation into cold clumps embedded in a hot tenuous gas via thermal instabilities. We show that cold clumps of ∼ 103 M⊙ are formed within ∼ 105 yrs. As a result, stars are produced along outflow's path, endowed with the outflow speed at their formation site. These HVSs travel through the galactic halo and eventually escape into the intergalactic medium. The expected instantaneous rate of star formation inside the outflow is ∼ 4 - 5 orders of magnitude greater than the average rate associated with previously proposed mechanisms for producing HVSs, such as the Hills mechanism and three-body interaction between a star and a black hole binary. We predict the spatial distribution of HVSs formed in AGN outflows for future observational probe.

  14. ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity.

    PubMed

    Milgrom, Y M; Ehler, L L; Boyer, P D

    1990-11-05

    The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.

  15. c-rel activates but v-rel suppresses transcription from kappa B sites.

    PubMed Central

    Inoue, J; Kerr, L D; Ransone, L J; Bengal, E; Hunter, T; Verma, I M

    1991-01-01

    We show that the product of the protooncogene c-rel is a constituent of an NF-kappa B-like complex that binds to the kappa B site originally identified in the enhancer of immunoglobulin kappa light chain gene. c-rel protein synthesized in bacteria binds to the kappa B site in a sequence-specific manner. The rel-kappa B complex can be disrupted by incubation with anti-rel antibodies. The rel protein can form oligomers. The c-rel protein can activate transcription from promoters containing kappa B sites; v-rel, on the other hand, suppresses the transcription of genes linked to kappa B sites. Thus, v-rel may interfere with the normal transcriptional machinery of the cell by acting as a dominant negative mutant. Images PMID:2023921

  16. Active-site monovalent cations revealed in a 1.55-Å-resolution hammerhead ribozyme structure.

    PubMed

    Anderson, Michael; Schultz, Eric P; Martick, Monika; Scott, William G

    2013-10-23

    We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na(+) ion binding in the ribozyme's active site. At least two such Na(+) ions are observed. The first Na(+) ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na(+) ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na(+), but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na(+) directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

    NASA Astrophysics Data System (ADS)

    Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

    1991-10-01

    Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site

  18. Microbial activities and dissolved organic matter dynamics in oil-contaminated surface seawater from the Deepwater Horizon oil spill site.

    PubMed

    Ziervogel, Kai; McKay, Luke; Rhodes, Benjamin; Osburn, Christopher L; Dickson-Brown, Jennifer; Arnosti, Carol; Teske, Andreas

    2012-01-01

    The Deepwater Horizon oil spill triggered a complex cascade of microbial responses that reshaped the dynamics of heterotrophic carbon degradation and the turnover of dissolved organic carbon (DOC) in oil contaminated waters. Our results from 21-day laboratory incubations in rotating glass bottles (roller bottles) demonstrate that microbial dynamics and carbon flux in oil-contaminated surface water sampled near the spill site two weeks after the onset of the blowout were greatly affected by activities of microbes associated with macroscopic oil aggregates. Roller bottles with oil-amended water showed rapid formation of oil aggregates that were similar in size and appearance compared to oil aggregates observed in surface waters near the spill site. Oil aggregates that formed in roller bottles were densely colonized by heterotrophic bacteria, exhibiting high rates of enzymatic activity (lipase hydrolysis) indicative of oil degradation. Ambient waters surrounding aggregates also showed enhanced microbial activities not directly associated with primary oil-degradation (β-glucosidase; peptidase), as well as a twofold increase in DOC. Concurrent changes in fluorescence properties of colored dissolved organic matter (CDOM) suggest an increase in oil-derived, aromatic hydrocarbons in the DOC pool. Thus our data indicate that oil aggregates mediate, by two distinct mechanisms, the transfer of hydrocarbons to the deep sea: a microbially-derived flux of oil-derived DOC from sinking oil aggregates into the ambient water column, and rapid sedimentation of the oil aggregates themselves, serving as vehicles for oily particulate matter as well as oil aggregate-associated microbial communities.

  19. Microbial Activities and Dissolved Organic Matter Dynamics in Oil-Contaminated Surface Seawater from the Deepwater Horizon Oil Spill Site

    PubMed Central

    Ziervogel, Kai; McKay, Luke; Rhodes, Benjamin; Osburn, Christopher L.; Dickson-Brown, Jennifer; Arnosti, Carol; Teske, Andreas

    2012-01-01

    The Deepwater Horizon oil spill triggered a complex cascade of microbial responses that reshaped the dynamics of heterotrophic carbon degradation and the turnover of dissolved organic carbon (DOC) in oil contaminated waters. Our results from 21-day laboratory incubations in rotating glass bottles (roller bottles) demonstrate that microbial dynamics and carbon flux in oil-contaminated surface water sampled near the spill site two weeks after the onset of the blowout were greatly affected by activities of microbes associated with macroscopic oil aggregates. Roller bottles with oil-amended water showed rapid formation of oil aggregates that were similar in size and appearance compared to oil aggregates observed in surface waters near the spill site. Oil aggregates that formed in roller bottles were densely colonized by heterotrophic bacteria, exhibiting high rates of enzymatic activity (lipase hydrolysis) indicative of oil degradation. Ambient waters surrounding aggregates also showed enhanced microbial activities not directly associated with primary oil-degradation (β-glucosidase; peptidase), as well as a twofold increase in DOC. Concurrent changes in fluorescence properties of colored dissolved organic matter (CDOM) suggest an increase in oil-derived, aromatic hydrocarbons in the DOC pool. Thus our data indicate that oil aggregates mediate, by two distinct mechanisms, the transfer of hydrocarbons to the deep sea: a microbially-derived flux of oil-derived DOC from sinking oil aggregates into the ambient water column, and rapid sedimentation of the oil aggregates themselves, serving as vehicles for oily particulate matter as well as oil aggregate-associated microbial communities. PMID:22509359

  20. Evidence for histidine in the active sites of ficin and stem-bromelain

    PubMed Central

    Husain, S. S.; Lowe, G.

    1968-01-01

    1. Ficin and stem-bromelain are irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. Evidence is presented that establishes that a histidine residue is within a 5Å locus of the active-site cysteine residue in both enzymes. The histidine residue in both enzymes is alkylated at N-1 by dibromoacetone. It is suggested that, as with papain, the thiol and imidazole groups act in concert in the hydrolysis of substrates by these enzymes. 2. The inhibition of thiol-subtilisin with 1,3-dibromoacetone is shown to be due to the alkylation of a cysteine residue only. PMID:5722692

  1. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenzamore » H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.« less

  2. Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

    PubMed

    Vickers, Chris J; Olsen, Christian A; Leman, Luke J; Ghadiri, M Reza

    2012-06-14

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

  3. Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

    PubMed

    Oyama, Takahiro; Yoshimori, Atsushi; Takahashi, Satoshi; Yamamoto, Tetsuya; Sato, Akira; Kamiya, Takanori; Abe, Hideaki; Abe, Takehiko; Tanuma, Sei-Ichi

    2017-07-01

    So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Acetylene hydratase: a non-redox enzyme with tungsten and iron-sulfur centers at the active site.

    PubMed

    Kroneck, Peter M H

    2016-03-01

    In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe-4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C2H2. To access the W-Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C2H2 binds directly to W. The architecture of the catalytic cavity, the specificity for C2H2 and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C2H2 are needed to present a conclusive answer.

  5. Wobble pairs of the HDV ribozyme play specific roles in stabilization of active site dynamics.

    PubMed

    Sripathi, Kamali N; Banáš, Pavel; Réblová, Kamila; Šponer, Jiří; Otyepka, Michal; Walter, Nils G

    2015-02-28

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5') hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5') general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5') hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs.

  6. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  7. Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

    PubMed Central

    Lucas, James E; Siegel, Justin B

    2015-01-01

    Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

  8. A gratuitous β-Lactamase inducer uncovers hidden active site dynamics of the Staphylococcus aureus BlaR1 sensor domain.

    PubMed

    Frederick, Thomas E; Peng, Jeffrey W

    2018-01-01

    Increasing evidence shows that active sites of proteins have non-trivial conformational dynamics. These dynamics include active site residues sampling different local conformations that allow for multiple, and possibly novel, inhibitor binding poses. Yet, active site dynamics garner only marginal attention in most inhibitor design efforts and exert little influence on synthesis strategies. This is partly because synthesis requires a level of atomic structural detail that is frequently missing in current characterizations of conformational dynamics. In particular, while the identity of the mobile protein residues may be clear, the specific conformations they sample remain obscure. Here, we show how an appropriate choice of ligand can significantly sharpen our abilities to describe the interconverting binding poses (conformations) of protein active sites. Specifically, we show how 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) exposes otherwise hidden dynamics of a protein active site that binds β-lactam antibiotics. When CBAP acylates (binds) the active site serine of the β-lactam sensor domain of BlaR1 (BlaRS), it shifts the time scale of the active site dynamics to the slow exchange regime. Slow exchange enables direct characterization of inter-converting protein and bound ligand conformations using NMR methods. These methods include chemical shift analysis, 2-d exchange spectroscopy, off-resonance ROESY of the bound ligand, and reduced spectral density mapping. The active site architecture of BlaRS is shared by many β-lactamases of therapeutic interest, suggesting CBAP could expose functional motions in other β-lactam binding proteins. More broadly, CBAP highlights the utility of identifying chemical probes common to structurally homologous proteins to better expose functional motions of active sites.

  9. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  10. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Paleomagnetism of the Chinle and Kayenta Formations, New Mexico and Arizona

    NASA Astrophysics Data System (ADS)

    Bazard, David R.; Butler, Robert F.

    1991-06-01

    Paleomagnetic data were obtained from 22 sites (6-10 samples/site) in the Upper Shale Member of the Chinle Formation, 43 sites in the Owl Rock Member of the Chinle Formation, and 35 sites in the Kayenta Formation. Thermal demagnetization and data analyses indicate that within-site dispersion is an important criterion for selecting sites which retain a high unblocking temperature characteristic remanent magnetization (ChRM). Site-mean directions define at least four antipodal polarity zones within each member/formation, suggesting the ChRM was acquired soon after deposition. Fifteen site-mean virtual geomagnetic poles (VGPs) from the Upper Shale Member of the Chinle Formation yield an early Norian paleomagnetic pole position of 57.4°N, 87.8°E (K = 60, A95 = 5.0°). Eighteen site-mean VGPs from the Owl Rock Member of the Chinle Formation yield a middle Norian paleomagnetic pole position of 56.5°N, 66.4°E (K = 183, A95 = 2.6°). Twenty-three site-mean VGPs from the Kayenta Formation yield a Pliensbachian pole position of 59.0°N, 66.6°E (K = 155, A95 = 2.4°). Combined with paleomagnetic poles from the Moenave Formation and the Shinarump Member of the Chinle Formation, these data record ˜30 m.y. of North American apparent polar wander (APW) within a regional stratigraphic succession. During the Camian and Norian stages of the Late Triassic, Chinle poles progress westward. During the Hettangian through Pliensbachian stages of the Early Jurassic, the pattern of APW changed to an eastward progression. Even after correction for 4° clockwise rotation of the Colorado Plateau, a sharp comer in the APW path (J1 cusp) is resolved near the pole from the Hettangian/Sinemurian (˜200 Ma) Moenave Formation (59.4°N, 59.2°E). Amongst other implications, the sharp change in the APW path at the J1 cusp implies an abrupt change from counterclockwise rotation of Pangea prior to 200 Ma to clockwise rotation thereafter.

  12. Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

    PubMed Central

    Wang, Lu; Fried, Stephen D.; Boxer, Steven G.; Markland, Thomas E.

    2014-01-01

    Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds. PMID:25503367

  13. Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

    PubMed

    Lenz, Stefan A P; Wetmore, Stacey D

    2016-02-09

    Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many

  14. Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

    PubMed

    Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

    2017-05-01

    RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.

  15. PKA activity is essential for relieving the suppression of hyphal growth and appressorium formation by MoSfl1 in Magnaporthe oryzae

    PubMed Central

    Li, Yang; Zhang, Xue

    2017-01-01

    In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and

  16. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

  17. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.

  18. Evaluation of physical activity web sites for use of behavior change theories.

    PubMed

    Doshi, Amol; Patrick, Kevin; Sallis, James F; Calfas, Karen

    2003-01-01

    Physical activity (PA) Web sites were assessed for their use of behavior change theories, including constructs of the health belief model, Transtheoretical Model, social cognitive theory, and the theory of reasoned action and planned behavior. An evaluation template for assessing PA Web sites was developed, and content validity and interrater reliability were demonstrated. Two independent raters evaluated 24 PA Web sites. Web sites varied widely in application of theory-based constructs, ranging from 5 to 48 on a 100-point scale. The most common intervention strategies were general information, social support, and realistic goal areas. Coverage of theory-based strategies was low, varying from 26% for social cognitive theory to 39% for health belief model. Overall, PA Web sites provided little assessment, feedback, or individually tailored assistance for users. They were unable to substantially tailor the on-line experience for users at different stages of change or different demographic characteristics.

  19. Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii.

    PubMed

    Pratap, Shivendra; Katiki, Madhusudhanarao; Gill, Preet; Kumar, Pravindra; Golemi-Kotra, Dasantila

    2016-01-01

    Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Dynamic asymmetry and the role of the conserved active-site thiol in rabbit muscle creatine kinase.

    PubMed

    Londergan, Casey H; Baskin, Rachel; Bischak, Connor G; Hoffman, Kevin W; Snead, David M; Reynoso, Christopher

    2015-01-13

    Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a

  1. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ...] Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf (OCS) Offshore... Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North Carolina... INFORMATION: Background: On December 13, 2012, BOEM published the Notice in the Federal Register (77 FR 74218...

  2. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sultatos, L.G.; Kaushik, R.

    2008-08-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, thesemore » changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.« less

  3. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations inmore » heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.« less

  4. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    NASA Astrophysics Data System (ADS)

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  5. Correlation between Surface Tension and Water Activity in New Particle Formation

    NASA Astrophysics Data System (ADS)

    Daskalakis, E.; Salameh, A.

    2016-12-01

    The impact of aerosol properties on cloud dynamics and the radiative balance of the atmosphere relies on the parametrizations of cloud droplet formation. Such parametrization is based on equilibrium thermodynamics proposed by Köhler in 1936. There is considerable debate in the literature on the importance of factors like the surface tension depression or the water activity decrease for the correct parametrization. To gain fundamental insight into New Particle Formation (NPF), or Cloud Condensation Nuclei (CCN) activation one has to study microscopic properties of aqueous droplets, involving surface and bulk dynamics. The surface tension of droplets can be associated with the effects from Organic Matter (OM), whereas the static dielectric constant of water reflects the structure and dynamics of ions within solutions and can present a measure of water activity. In this study we employ Molecular Dynamics Simulations on aquatic droplets that contain surface active OM (acetaldehyde, methylglyoxal) and salts. We give insight into the dynamics of aquatic droplets with radials of 3.6nm at a level of detail that is not accessible experimentally (J. Phys. Chem. C 2016, 120:11508). We propose that as the surface tension of an aquatic droplet is decreased in the presence of surface-active OM, the water activity is affected as well. This is due to the fact that the water dipoles are oriented based on the salt morphology within the droplet. We suggest that the surface tension depression can be accompanied by the water activity change. This can be associated with the possible effects of surface-active species in terms of salt morphology transitions within an aerosol at the NPF and early particle growth time scales. Based on this study, surface-active OM seems important in controlling (a) the salt morphology transitions within a nucleus during NPF and particle growth and (b) a correlation between surface activity and water activity of ionic aquatic droplets. The latter

  6. Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes

    PubMed Central

    Sarker, Rafiquel; Cha, Boyoung; Kovbasnjuk, Olga; Cole, Robert; Gabelli, Sandra; Tse, Chung Ming; Donowitz, Mark

    2017-01-01

    Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity—specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity—specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity. PMID:28495796

  7. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

    PubMed Central

    Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-01-01

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  8. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  9. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

    DOE PAGES

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

    2014-12-31

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

  10. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

  11. Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

    PubMed Central

    Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

    2016-01-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  12. Propofol, more than halothane, depresses electroencephalographic activation resulting from electrical stimulation in reticular formation.

    PubMed

    Antognini, J F; Bravo, E; Atherley, R; Carstens, E

    2006-09-01

    Halothane and propofol depress the central nervous system, and this is partly manifested by a decrease in electroencephalographic (EEG) activity. Little work has been performed to determine the differences between these anesthetics with regard to their effects on evoked EEG activity. We examined the effects of halothane and propofol on EEG responses to electrical stimulation of the reticular formation. Rats (n= 12) were anesthetized with either halothane or propofol, and EEG responses were recorded before and after electrical stimulation of the reticular formation. Two anesthetic concentrations were used (0.8 and 1.2 times the amount needed to prevent gross, purposeful movement in response to supramaximal noxious stimulation), and both anesthetics were studied in each rat using a cross-over design. Electrical stimulation in the reticular formation increased the spectral edge (SEF) and median edge (MEF) frequencies by approximately 1-2 Hz during halothane anesthesia at low and high concentrations. During propofol anesthesia, MEF increased at the low propofol infusion rate, but SEF was unaffected. At the high propofol infusion rate, SEF and MEF decreased following electrical stimulation in the reticular formation. At immobilizing concentrations, propofol produces a larger decrease than halothane in EEG responses to reticular formation stimulation, consistent with propofol having a more profound depressant effect on cortical and subcortical structures.

  13. A format for the interchange of scheduling models

    NASA Technical Reports Server (NTRS)

    Jaap, John P.; Davis, Elizabeth K.

    1994-01-01

    In recent years a variety of space-activity schedulers have been developed within the aerospace community. Space-activity schedulers are characterized by their need to handle large numbers of activities which are time-window constrained and make high demands on many scarce resources, but are minimally constrained by predecessor/successor requirements or critical paths. Two needs to exchange data between these schedulers have materialized. First, there is significant interest in comparing and evaluating the different scheduling engines to ensure that the best technology is applied to each scheduling endeavor. Second, there is a developing requirement to divide a single scheduling task among different sites, each using a different scheduler. In fact, the scheduling task for International Space Station Alpha (ISSA) will be distributed among NASA centers and among the international partners. The format used to interchange scheduling data for ISSA will likely use a growth version of the format discussed in this paper. The model interchange format (or MIF, pronounced as one syllable) discussed in this paper is a robust solution to the need to interchange scheduling requirements for space activities. It is highly extensible, human-readable, and can be generated or edited with common text editors. It also serves well the need to support a 'benchmark' data case which can be delivered on any computer platform.

  14. DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

    PubMed

    Usui, Kenji; Okada, Arisa; Sakashita, Shungo; Shimooka, Masayuki; Tsuruoka, Takaaki; Nakano, Shu-Ichi; Miyoshi, Daisuke; Mashima, Tsukasa; Katahira, Masato; Hamada, Yoshio

    2017-11-16

    The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca 2+ , and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

  15. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 andmore » A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their

  16. The Suppression of Star Formation by Powerful Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Dwek, E.

    2012-01-01

    The old, red stars that constitute the bulges of galaxies, and the massive black holes at their centres, are the relics of a period in cosmic history when galaxies formed stars at remarkable rates and active galactic nuclei (AGN) shone brightly as a result of accretion onto black holes. It is widely suspected, but unproved, that the tight corre1ation between the mass of the black hole and the mas. of the stellar bulge results from the AGN quenching the surrounding star formation as it approaches its peak luminosity. X-rays trace emission from AGN unambiguously, whereas powerful star-forming ga1axies are usually dust-obscured and are brightest at infrared and submillimeter wavelengths. Here we report submillimetre and X-ray observations that show that rapid star formation was common in the host galaxies of AGN when the Universe was 2-6 billion years old, but that the most vigorous star formation is not observed around black holes above an X-ray luminosity of 10(exp 44) ergs per second. This suppression of star formation in the host galaxy of a powerful AGN is a key prediction of models in which the AGN drives an outflow, expe11ing the interstellar medium of its host and transforming the galaxy's properties in a brief period of cosmic time.

  17. Development of soil chemical and biological properties in the initial stages of post-mining deposition sites.

    PubMed

    Monokrousos, Nikolaos; Boutsis, George; Diamantopoulos, John D

    2014-12-01

    The aim of this study was to assess the seasonal development of the physicochemical (pH, organic C, organic N, extractable P, Ca(2+), Mg(2+)) and biological soil properties (microbial biomass, activities of urease, dehydrogenase and alkaline phosphatase) of the topsoil of mine deposition sites that differed based on the material used exclusively for their creation: (a) marlstones, (b) red-grey formations (RGF), and (c) fly ash (FA), during the first year after their creation. Our hypothesis was that all deposition sites, regardless the material they consist of, present equal opportunities for the establishment of spontaneous vegetation. All macronutrients concentrations (P, Ca(2+), and Mg(2+)) remained constant with time and were found to be higher in the FA sites. Organic C, organic N, all enzyme activities, and microbial biomass were higher in the RGF and marl depositions, with marl sites presenting the highest values. All values of biological variables, with the exception of alkaline phosphatase, increased with time. The alkaline environment along with the slow improvement in soil biological properties of the FA sites seemed to present the most unfavorable conditions for spontaneous vegetation growth. On the contrary, the other two spoil materials presented significant improvement in the initial stages of soil formation in terms of soil functionality.

  18. Opportunities for increasing CO 2 storage in deep, saline formations by active reservoir management and treatment of extracted formation water: Case study at the GreenGen IGCC facility, Tianjin, PR China

    DOE PAGES

    Ziemkiewicz, Paul; Stauffer, Philip H.; Sullivan-Graham, Jeri; ...

    2016-08-04

    Carbon capture, utilization and storage (CCUS) seeks beneficial applications for CO 2 recovered from fossil fuel combustion. This study evaluated the potential for removing formation water to create additional storage capacity for CO 2, while simultaneously treating the produced water for beneficial use. Furthermore, the process would control pressures within the target formation, lessen the risk of caprock failure, and better control the movement of CO 2 within that formation. The project plans to highlight the method of using individual wells to produce formation water prior to injecting CO 2 as an efficient means of managing reservoir pressure. Because themore » pressure drawdown resulting from pre-injection formation water production will inversely correlate with pressure buildup resulting from CO 2 injection, it can be proactively used to estimate CO 2 storage capacity and to plan well-field operations. The project studied the GreenGen site in Tianjin, China where Huaneng Corporation is capturing CO 2 at a coal fired IGCC power plant. Known as the Tianjin Enhanced Water Recovery (EWR) project, local rock units were evaluated for CO 2 storage potential and produced water treatment options were then developed. Average treatment cost for produced water with a cooling water treatment goal ranged from 2.27 to 2.96 US$/m 3 (recovery 95.25%), and for a boiler water treatment goal ranged from 2.37 to 3.18 US$/m 3 (recovery 92.78%). Importance analysis indicated that water quality parameters and transportation are significant cost factors as the injection-extraction system is managed over time. Our study found that in a broad sense, active reservoir management in the context of CCUS/EWR is technically feasible. In addition, criteria for evaluating suitable vs. unsuitable reservoir properties, reservoir storage (caprock) integrity, a recommended injection/withdrawal strategy and cost estimates for water treatment and reservoir management are proposed.« less

  19. Opportunities for increasing CO 2 storage in deep, saline formations by active reservoir management and treatment of extracted formation water: Case study at the GreenGen IGCC facility, Tianjin, PR China

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ziemkiewicz, Paul; Stauffer, Philip H.; Sullivan-Graham, Jeri

    Carbon capture, utilization and storage (CCUS) seeks beneficial applications for CO 2 recovered from fossil fuel combustion. This study evaluated the potential for removing formation water to create additional storage capacity for CO 2, while simultaneously treating the produced water for beneficial use. Furthermore, the process would control pressures within the target formation, lessen the risk of caprock failure, and better control the movement of CO 2 within that formation. The project plans to highlight the method of using individual wells to produce formation water prior to injecting CO 2 as an efficient means of managing reservoir pressure. Because themore » pressure drawdown resulting from pre-injection formation water production will inversely correlate with pressure buildup resulting from CO 2 injection, it can be proactively used to estimate CO 2 storage capacity and to plan well-field operations. The project studied the GreenGen site in Tianjin, China where Huaneng Corporation is capturing CO 2 at a coal fired IGCC power plant. Known as the Tianjin Enhanced Water Recovery (EWR) project, local rock units were evaluated for CO 2 storage potential and produced water treatment options were then developed. Average treatment cost for produced water with a cooling water treatment goal ranged from 2.27 to 2.96 US$/m 3 (recovery 95.25%), and for a boiler water treatment goal ranged from 2.37 to 3.18 US$/m 3 (recovery 92.78%). Importance analysis indicated that water quality parameters and transportation are significant cost factors as the injection-extraction system is managed over time. Our study found that in a broad sense, active reservoir management in the context of CCUS/EWR is technically feasible. In addition, criteria for evaluating suitable vs. unsuitable reservoir properties, reservoir storage (caprock) integrity, a recommended injection/withdrawal strategy and cost estimates for water treatment and reservoir management are proposed.« less

  20. Coordination of FocA and Pyruvate Formate-Lyase Synthesis in Escherichia coli Demonstrates Preferential Translocation of Formate over Other Mixed-Acid Fermentation Products

    PubMed Central

    Beyer, Lydia; Doberenz, Claudia; Falke, Dörte; Hunger, Doreen; Suppmann, Bernhard

    2013-01-01

    Enterobacteria such as Escherichia coli generate formate, lactate, acetate, and succinate as major acidic fermentation products. Accumulation of these products in the cytoplasm would lead to uncoupling of the membrane potential, and therefore they must be either metabolized rapidly or exported from the cell. E. coli has three membrane-localized formate dehydrogenases (FDHs) that oxidize formate. Two of these have their respective active sites facing the periplasm, and the other is in the cytoplasm. The bidirectional FocA channel translocates formate across the membrane delivering substrate to these FDHs. FocA synthesis is tightly coupled to synthesis of pyruvate formate-lyase (PflB), which generates formate. In this study, we analyze the consequences on the fermentation product spectrum of altering FocA levels, uncoupling FocA from PflB synthesis or blocking formate metabolism. Changing the focA translation initiation codon from GUG to AUG resulted in a 20-fold increase in FocA during fermentation and an ∼3-fold increase in PflB. Nevertheless, the fermentation product spectrum throughout the growth phase remained similar to that of the wild type. Formate, acetate, and succinate were exported, but only formate was reimported by these cells. Lactate accumulated in the growth medium only in mutants lacking FocA, despite retaining active PflB, or when formate could not be metabolized intracellularly. Together, these results indicate that FocA has a strong preference for formate as a substrate in vivo and not other acidic fermentation products. The tight coupling between FocA and PflB synthesis ensures adequate substrate delivery to the appropriate FDH. PMID:23335413